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Vitamin D accelerates resolution of inflammatory responses during tuberculosis treatmentAnna K. Coussensa, Robert J. Wilkinsona,b, Yasmeen Hanifac, Vladyslav Nikolayevskyyd, Paul T. Elkingtone,Kamrul Islamc, Peter M. Timmsf, Timothy R. Ventonf, Graham H. Bothamleyf, Geoffrey E. Packeg,Mathina Darmalingamh, Robert N. Davidsoni, Heather J. Milburnj, Lucy V. Bakerk, Richard D. Barkerl,Charles A. Meinm, Leena Bhaw-Rosunm, Rosamond Nuamahm, Douglas B. Younga, Francis A. Drobniewskid,Christopher J. Griffithsc, and Adrian R. Martineauc,a,b,1 aDivision of Mycobacterial Research, Medical Research Council National Institute for Medical Research, London NW7 1AA, United Kingdom; bDivision ofMedicine, Imperial College London, London W2 1PG, United Kingdom; cBlizard Institute, Barts and The London School of Medicine and Dentistry, Queen MaryUniversity of London, London E1 2AB, United Kingdom; dHealth Protection Agency National Mycobacterium Reference Laboratory, Barts and The LondonSchool of Medicine and Dentistry, London E1 2AT, United Kingdom; eDepartment of Infectious Diseases and Immunity, Imperial College London, London W120NN, United Kingdom; fHomerton University Hospital, London E9 6SR, United Kingdom; gNewham Chest Clinic, Forest Gate, London E7 8QP, United Kingdom; hDepartment of Respiratory Medicine, Whipps Cross University Hospital, London E11 1NR, United Kingdom; iTuberculosis Clinic, Northwick Park Hospital,Harrow HA1 3UJ, United Kingdom; jDepartment of Respiratory Medicine, Guy's and St Thomas' Hospitals, London SE1 9RT, United Kingdom; kDepartment ofRespiratory Medicine, Lewisham Hospital, London SE13 6LH, United Kingdom; lDepartment of Respiratory Medicine, Kings College Hospital, London SE5 9RS,United Kingdom; and mGenome Centre, Barts and The London School of Medicine, Queen Mary University of London, London EC1M 6BQ, United Kingdom Edited* by Barry R. Bloom, Harvard School of Public Health, Boston, MA, and approved July 25, 2012 (received for review January 18, 2012) Calcidiol, the major circulating metabolite of vitamin D, supports pathogen-associated ligands (11, 12). Extrarenal generation of induction of pleiotropic antimicrobial responses in vitro. Vitamin D calcitriol is dependent on the availability of its precursor calcidiol, supplementation elevates circulating calcidiol concentrations, and the major circulating vitamin D metabolite that supports in- thus has a potential role in the prevention and treatment of in- duction of antimicrobial responses in vitro (11, 13) and the con- fection. The immunomodulatory effects of administering vitamin D centrations of which are often low in patients with pulmonary to humans with an infectious disease have not previously been re- infection (14–16). Vitamin D supplementation elevates circulat- ported. To characterize these effects, we conducted a detailed ing calcidiol concentrations, and may therefore enhance response longitudinal study of circulating and antigen-stimulated immune to antimicrobial therapy for respiratory infections. However, responses in ninety-five patients receiving antimicrobial therapy for the effects of in vivo vitamin D supplementation on immune re-sponses in humans with an infectious disease have not previously pulmonary tuberculosis who were randomized to receive adjunctive been described.
high-dose vitamin D or placebo in a clinical trial, and who fulfilled Vitamin D was used to treat tuberculosis in the preantibiotic criteria for per-protocol analysis. Vitamin D supplementation accel- era (17), and vitamin D supplementation has been shown to erated sputum smear conversion and enhanced treatment-induced enhance healthy tuberculosis contacts' immunity to mycobacteria resolution of lymphopaenia, monocytosis, hypercytokinaemia, and (18). These observations prompted us to conduct a randomized hyperchemokinaemia. Administration of vitamin D also suppressed controlled trial evaluating the influence of adjunctive high-dose antigen-stimulated proinflammatory cytokine responses, but atten- vitamin D on time to bacterial clearance in patients receiving uated the suppressive effect of antimicrobial therapy on antigen- antimicrobial therapy for smear-positive pulmonary tuberculosis stimulated secretion of IL-4, CC chemokine ligand 5, and IFN-α. We (19). We now present results of a detailed analysis of longitu- demonstrate a previously unappreciated role for vitamin D supple- dinal changes in the immune response in trial participants during mentation in accelerating resolution of inflammatory responses dur- the 8-wk course of intensive-phase antituberculous therapy.
ing tuberculosis treatment. Our findings suggest a potential role for Initially we describe the effects of antituberculous therapy alone adjunctive vitamin D supplementation in the treatment of pulmo- on circulating and antigen-stimulated immune responses, using nary infections to accelerate resolution of inflammatory responses samples from patients randomized to the placebo arm of the associated with increased risk of mortality.
trial. Subsequently we proceed to characterize the effects of vi-tamin D supplementation using samples from those randomized adjunctive therapy immunomodulation antimicrobial peptides to the intervention arm of the study, showing that administrationof adjunctive vitamin D exerts pleiotropic immunomodulatory effects in patients with pulmonary tuberculosis.
espite the widespread availability of antimicrobials, bacterialrespiratory infections remain a major global cause of death (1). Mortality is associated with infection with antibiotic-resistantorganisms (2, 3) and with failure to resolve immunopathological Effect of Antimicrobials on Circulating Responses. Determination of inflammatory responses (4–6). Immunomodulatory agents that the immunomodulatory effects of vitamin D supplementationnecessitated an initial, comprehensive characterization of the augment antimicrobial immune responses and accelerate reso- changes in immune responses induced by antituberculous therapy lution of pulmonary inflammation could be used as adjuncts to alone. To this end, 42 soluble factors and 14 hematological antimicrobial therapy to improve treatment outcomes (7).
Calcitriol, the active metabolite of vitamin D, induces innate antimicrobial responses and suppresses proinflammatory cytokineresponses in vitro (8). Calcitriol's antimicrobial activity is mediated Author contributions: C.J.G. and A.R.M. designed research; A.K.C., R.J.W., Y.H., V.N., P.T.E.,K.I., P.M.T., T.R.V., G.H.B., G.E.P., M.D., R.N.D., H.J.M., L.V.B., R.D.B., C.A.M., L.B.-R., R.N., via induction of reactive nitrogen intermediates, reactive oxygen D.B.Y., F.A.D., C.J.G., and A.R.M. performed research; A.K.C. and A.R.M. analyzed data; intermediates, antimicrobial peptides, and autophagy (9). Calci- and A.K.C. and A.R.M. wrote the paper.
triol also modulates adaptive responses, both indirectly (by sup- Conflict of interest statement: Merck Serono donated €7,000 to Queen Mary University of pression of MHC class II expression and IL-12 secretion by London in 2010 to support an academic meeting entitled "Vitamin D: Mechanisms of antigen-presenting cells) and directly [by suppressing secretion of Action in Health and Disease"; this meeting was convened by C.J.G. and A.R.M.
IFN-γ and IL-2 from CD4+ T-helper type 1 (Th1) cells] (10).
*This Direct Submission article had a prearranged editor.
Calcitriol is synthesized by the vitamin D 1-α hydroxylase enzyme, 1To whom correspondence should be addressed. E-mail: the expression of which is up-regulated in leukocytes and pul- This article contains supporting information online at monary epithelium following ligation of Toll-like receptors by PNAS Early Edition 1 of 6 parameters, detailed in Materials and Methods, were measured in Effect of Antimicrobials on Antigen-Stimulated Responses. Whole- samples of serum, plasma, and whole blood taken from 51 blood samples taken from 28 patients randomized to the placebo patients randomized to the placebo arm of the trial at 0, 2, 4, 6, arm of the trial were stimulated ex vivo with a panel of mycobac- and 8 wk of treatment (for trial profile, see for baseline terial antigens, and the concentration of IFN-γ in supernatants of characteristics, see Parameters were selected on the baseline samples was compared between stimuli. Of the MTB- basis that they played a role in host defense against Mycobacte- specific antigens tested, recombinant early-secreted antigenic tar- rium tuberculosis (MTB) (20) or that they were biomarkers of get 6 kDa (rESAT-6) and recombinant culture filtrate protein 10 treatment response (21). Median serum concentrations of seven kDa (rCFP-10) induced the greatest IFN-γ responses ). We soluble factors [IL-2, IL-5, IL-13, IL-17, TNF, FGF-β, and matrix therefore proceeded to assay concentrations of the 39 soluble metalloproteinase-7 (MMP-7)] were below the limit of detection factors listed in Materials and Methods in supernatants of whole at baseline, and these were excluded from statistical analyses. The blood stimulated with these two antigens at 0, 2, 4, 6, and 8 wk of remaining 49 parameters were assessed using principal compo- treatment. Median concentrations of six soluble factors (IL-2, IL- nent analysis (PCA), a well-established mathematical technique 5, IL-13, EGF, FGF-β, and MMP-7) were below the limit of de- for reducing the dimensionality of complex datasets by trans- tection in these samples, and these parameters were therefore forming the data to a new coordinate system. The first three excluded from statistical analyses. The remaining 33 parameters coordinates (principal components) are represented as a 3D plot.
underwent PCA transformation and rank regression analysis. The The first principal component accounts for as much of the vari- resultant PCA plots for rESAT-6–and rCFP-10–stimulated ability in the data as possible, and each succeeding component responses were similar to each other: samples has the highest variance possible under the constraint that it is converged from a loosely clustered pattern at baseline toward uncorrelated with preceding components. This method allows a more tightly clustered pattern at 4 wk, and then back to a more visualization of the differences between patient samples and loosely clustered pattern at 8 wk, changes confirmed as being analytes within complex datasets (22). The resultant PCA plot statistically significant by analysis of the sums of Euclidean dis- showed that baseline samples were less tightly tances at these time points (P < 0.0001) (Twenty- clustered than follow-up samples. Comparison of the median sum seven antigen-stimulated parameters contributed to the pattern of of Euclidean distances between points in the PCA plot at baseline response to antituberculous therapy (). All analytes whose vs. 8 wk confirmed that this convergence was statistically signifi- concentration changed significantly during the course of intensive cant (P < 0.0001) (), indicating that patients had a rela- phase antituberculous therapy showed a decrease in secretion over tively heterogeneous circulating immunological profile at baseline time. Of note, IFN-γ was among the analytes whose antigen- that became more homogenous as treatment progressed.
stimulated concentration did not change significantly during the Rank-regression analysis (23) was applied to PCA-transformed course of antituberculous therapy, even when corrected for data to identify parameters whose concentration changed signifi- changes in lymphocyte count ).
cantly over time. presents details of the 42 circulating To determine whether changes in antigen-stimulated immune immunological parameters so identified. Of the hematological responses corresponded to changes in whole-blood cellular parameters investigated, platelet count, neutrophil count, and composition during antituberculous therapy, network PCA was monocyte count decreased during the course of treatment (P ≤ applied to the antigen-stimulated analytes listed in 0.0018), but lymphocyte count and eosinophil count both in- together with cell-count data obtained for the relevant samples creased (P ≤ 0.0019). Increases were also seen in hemoglobin before antigenic stimulation. Similar PCA networks were iden- concentration and red blood cell parameters (P ≤ 0.015), reflect- tified for rCFP-10– and rESAT-6–stimulated responses ( ing resolution of microcytic anemia as treatment progressed.
). Platelets, CCL5, IL-4, G-CSF, and CCL11 were Decreases in erythrocyte sedimentation rate (ESR) and C-reactive connected in both plots, replicating the platelet–CCL5 connec- protein (CRP) (P ≤ 1.24 × 10−12) and an increase in serum al- tion observed in the analysis of circulating parameters ( bumin concentration (P = 5.17 × 10−21) were also seen, indicating ). Neutrophils occupied a similar space to neutrophil gran- resolution of the acute-phase response. These changes were ac- ule-associated proteins MMP-9 and NGAL. IL-7 clustered with companied by a decrease in circulating concentrations of all another angiogenic factor, VEGF, and Th1 cytokines. Lympho- cytokines, chemokines, antimicrobial peptides (AMP), MMP, and cytes were not connected directly to any cytokines stimulated by angiogenic factors identified (P ≤ 0.0240), except for CC chemo- rESAT-6 or rCFP-10.
kine ligand 2 (CCL2) and MMP-2, the concentrations of whichincreased during the course of treatment (P ≤ 0.0035).
Effects of Vitamin D on Circulating Responses. We have previously To investigate the relationship between changes in cell counts reported results of the intention-to-treat analysis of study data, and circulating concentrations of inflammatory mediators observed indicating that administration of adjunctive vitamin D was asso- during treatment, a PCA network was created for the parameters ciated with a trend toward faster sputum culture conversion (P = listed in . This network connected each analyte to one 0.14) (19). We repeated this analysis in the subgroup of 95 par- other analyte with which it shared the most similar pattern of ticipants fulfilling per-protocol analysis criteria, adjusting for fac- change over time; the distance between analytes in the network tors previously shown to influence time-to-sputum conversion in represents their Pearson correlation coefficients. Seven distinct this dataset (19) (age, ethnicity, baseline sputum smear, neutrophil clusters were identified ). For parameters whose values count, and presence or absence of cavitation on baseline chest fell during treatment, the tightest cluster incorporated three MMP radiograph). Median time to sputum culture conversion in this (MMP-1, MMP-8, and MMP-9) with three AMP [human neutro- subset of patients was 35 d in the intervention group and 46.5 d in phil peptides (HNP) 1–3, neutrophil gelatinase-associated lipocalin the control group (hazard ratio 1.27, 95% confidence interval (NGAL), and cathelicidin (LL-37)]; this cluster was close to neu- 0.76–2.13, P = 0.36), and median time to sputum smear conver- trophils, CXC chemokine ligand 8 (CXCL8), and prostaglandin E2 sion in the intervention arm was significantly shorter than that in (PGE2). Neutrophils were linked to monocytes and CRP, which in the control arm (23 vs. 36 d; hazard ratio 1.69, 95% confidence turn was linked to IL-6 and ESR. Platelet count and CCL5 formed interval 1.02–2.79, P = 0.04) (Fig. 1A).
a distinct grouping, and the other IFN-γ–stimulated chemokines, To determine whether the effect of vitamin D on time to sputum CXCL9 and CXCL10, were linked to each other and to IFN-γ, clearance in the per-protocol subgroup was associated with im- which was linked to IL-6. The angiogenic factors, EGF, hepatocyte munomodulatory activity, we compared the effect of antituber- growth factor (HGF), and VEGF were linked to each other and to culous therapy on the circulating immunological parameters IL-7, IL-10, IL-15, and soluble IL-2 receptor (IL-2R). For investigated above in 51 patients randomized to the placebo arm parameters increasing during treatment, one network incorporated of the trial vs. 44 patients randomized to receive adjunctive vitamin red blood cell parameters, albumin, and MMP-2, and another D using PCA and rank regression on the interaction term "treat- linked lymphocyte and eosinophil counts.
ment duration*allocation." The PCA plot is shown in .
Coussens et al.
Kinetics of circulating and antigen-stimulated immune responses during the course of antituberculous therapy in the presence vs. the absence of ad- junctive vitamin D. Vitamin D accelerated sputum smear conversion in patients fulfilling per-protocol analysis criteria (A). Monocyte counts fell more quickly (B)and lymphocyte counts rose more quickly (C) among patients in the intervention arm of the trial. Vitamin D also accelerated treatment-induced decreases in ESR(D), circulating concentrations of CXCL9 (E), CXCL10 (F), MMP-9 (G), and LL-37 (H) and rCFP-10–stimulated supernatant concentrations of IL-1RA (I), IL-6 (J),IL-12p40/p70 (K), and TNF (L). Means ± SEM at 0, 2, 4, 6, and 8 wk of treatment are presented. Dotted lines, placebo arm; solid lines, vitamin D arm.
The 17 circulating parameters identified as being significantly receptor (VDR), such that vitamin D hastened sputum culture affected by vitamin D are detailed in All of these conversion in patients with the tt genotype, but not in those with parameters were also significantly affected by intensive-phase Tt or TT genotypes (Fig. 2 A–C) (19). To investigate whether the antituberculous therapy: in every case, vitamin D accelerated the effects of vitamin D on circulating immune responses were also effect of antituberculous therapy. The analyte most affected by restricted to patients with this genotype, we stratified analysis of vitamin D was the chemokine CXCL9, whose serum concentration the effects of vitamin D on 8-wk values of analytes listed in decreased significantly faster in patients randomized to receive according to TaqI genotype; patients with the tt genotype vitamin D vs. placebo (P = 5.92 × 10−12). Serum concentrations were not included in this analysis because of small numbers with of three other chemokines (CXCL10, CCL3, and CCL5) also fell immunological data available (n = 7). In patients with the TT more rapidly in patients randomized to vitamin D vs. placebo (P ≤ genotype, vitamin D supplementation significantly reduced 8-wk 0.0164), as did IFN-γ (P = 0.0012). Monocyte counts fell more circulating concentrations of CRP, CXCL9, CXCL10, NGAL, rapidly (P = 0.0003) and lymphocyte counts rose more rapidly and LL-37, and in those with the Tt genotype, vitamin D sig- (P = 0.0364) among patients receiving vitamin D. Neutrophil nificantly reduced 8-wk circulating concentrations of CXCL9 and counts were not significantly affected by allocation, but plasma IL-10 (Fig. 2 D–I) (P ≤ 0.04). Our finding that vitamin D sup- concentrations of neutrophil-associated AMP (LL-37, HNP1–3, plementation modulated immune responses in patients with the and NGAL) and MMP-9 fell more quickly in patients receiving Tt and TT genotypes indicates that immunomodulatory effects of adjunctive vitamin D (P ≤ 0.0112). Administration of vitamin D vitamin D are not restricted to individuals with the tt genotype of also induced a more rapid drop in ESR and serum CRP concen- the TaqI VDR polymorphism.
tration (P ≤ 0.0072), indicating accelerated resolution of theacute-phase response.
Effects of Vitamin D on Antigen-Stimulated Responses. We next in- Network PCA indicated that the accelerated fall in monocyte vestigated the effect of vitamin D supplementation on antigen- count in patients receiving vitamin D was linked to a rise in lym- stimulated responses using PCA and rank regression interaction phocyte count and a decrease in ESR and serum CRP concentration analysis on 33 analytes detailed above for 19 patients allocated to The IFN-γ–inducible chemokines CXCL9 and CXCL10 vitamin D vs. 28 patients allocated to placebo. The PCA plots were linked to IFN-γ, IL-2R, IL-10, and CCL3. NGAL, HNP1–3, generated for responses to rESAT-6 and rCFP-10 were similar to and MMP-9 formed another cluster, which was linked to LL-37 and each other, and samples from patients allocated to vitamin D vs.
PGE2. The kinetics of change in a representative group of circu- placebo were clearly separated (). Vitamin D lating immunological parameters over the course of intensive-phase supplementation influenced supernatant concentrations of seven therapy in vitamin D vs. placebo arms are presented in Fig. 1 B–H.
analytes in both rESAT-6– and rCFP-10–stimulated whole blood We have previously reported that the effect of vitamin D on ); notably, IFN-γ was not among them (Vi- time to sputum culture conversion in the intention-to-treat tamin D enhanced the suppressive effect of antimicrobial ther- analysis was modified by the TaqI genotype of the vitamin D apy on secretion of IL-1 receptor antagonist (IL-1RA), IL-6, IL- Coussens et al.
PNAS Early Edition 3 of 6 Immunomodulatory actions of vitamin D are not restricted to individuals with the tt genotype of the TaqI vitamin D receptor polymorphism. Vitamin D supplementation accelerates sputum culture conversion in patients with the tt genotype of the TaqI vitamin D receptor polymorphism (A), but not in thosewith the Tt (B) or TT (C) genotypes (Pinteraction = 0.03). Vitamin D, solid line; placebo, dotted line. In contrast, the immunomodulatory actions of vitamin D arenot restricted to those with the tt genotype of the TaqI polymorphism. In patients with the TT genotype, vitamin D supplementation significantly reduced 8-wk circulating concentrations of CRP (D), CXCL9 (E), CXCL10 (F), NGAL (G), and LL-37 (H); in patients with the Tt genotype, statistically significant reductions in8-wk serum concentrations of CXCL9 (E) and IL-10 (I) were also seen in patients randomized to vitamin D (Vit D) vs. Placebo (Plac). Data for patients with the ttgenotype are not presented because of small numbers entering per-protocol analysis. Line at median; TT placebo (□) n = 23, TT vitamin D (■) n = 22, Ttplacebo (○) n = 21, Tt vitamin D () n = 19. LOD, limit of detection.
12, and TNF (P ≤ 0.0437), and attenuated treatment-induced during treatment of an infectious disease. In patients taking reductions in secretion of IL-4, CCL5, and IFN-α (P ≤ 0.0323).
antimicrobial therapy for smear-positive pulmonary tuberculo- Network PCA showed that IL-12, TNF, and IL-1RA were linked, sis, adjunctive vitamin D accelerated sputum smear conversion, but IL-6 was connected to monocytes directly or via VEGF, and augmented treatment-induced increases in lymphocyte count, CCL5 and IL-4 were tightly clustered and linked to IFN-α for and enhanced the suppressive effect of treatment on monocyte both antigens (Vitamin D significantly reduced count, inflammatory markers, and circulating concentrations of antigen-stimulated secretion of IL-12, TNF, IL-1RA, IL-6, chemokines, AMPs, and MMP-9. Administration of vitamin D CXCL10, CCL3, CCL4, and VEGF (Fig. 1 I–L) and enhanced also enhanced treatment-induced suppression of antigen-stimulated CCL5, CCL11, IL-4, and IFN-α secretion.
Th1 cytokine responses, but attenuated treatment-induced sup-pression of antigen-stimulated IL-4, CCL5, and IFN-α secretion.
Among 51 patients randomized to receive antituberculous Our study represents the most detailed characterization of the therapy plus placebo, we observed an increase in circulating lym- effects of antituberculous therapy on the immune response con- phocyte counts and a reduction in circulating neutrophil counts, ducted to date, and is unique in being a clinical investigation into the monocyte counts, and concentrations of IFN-inducible parame- immunomodulatory actions of in vivo vitamin D supplementation ters following initiation of antituberculous therapy, consistent with Coussens et al.
previous reports (24–26). These changes were associated with Although many of the immunomodulatory effects of in vivo decreases in circulating concentrations of lymphocyte chemo- vitamin D supplementation that we observed were in keeping attractants CXCL9 and CXCL10, and an increase in the monocyte with the in vitro actions of calcitriol, there were two exceptions: chemoattractant CCL2; they may therefore reflect reduced re- calcitriol has been reported to induce IL-10 (36) and the anti- cruitment of lymphocytes and increased recruitment of monocytes microbial peptides LL-37 and NGAL (40, 41) in vitro, but we to the lung. In keeping with this hypothesis, the proportion of found that in vivo vitamin D supplementation suppressed cir- macrophages in sputum of tuberculosis patients has been reported culating concentrations of IL-10, LL-37, and NGAL. All three of to increase as treatment progresses (27). Interestingly, increases in these markers are suppressed by antituberculous therapy alone circulating lymphocyte count following initiation of antituberculous and the fact their concentration fell more quickly therapy were not associated with any change in antigen-stimu- among patients in the intervention arm of the study may arise as lated production of IFN-γ as treatment progressed. In contrast, an indirect consequence of enhanced microbial killing in patients antigen-stimulated production of CCL5, IL-4, G-CSF, IFN-α, and receiving vitamin D. Alternatively, this observation may repre- CXCL10 were greatly decreased over the course of intensive- sent a direct suppressive effect of vitamin D on release of these phase therapy. Antigen-stimulated CXCL10 responses have been mediators into the circulation from neutrophil granules.
reported to be more sensitive than IFN-γ for the diagnosis of Interestingly, and in contrast to the effects of vitamin D sup- active tuberculosis (28), and these data suggest that this panel of plementation on sputum clearance that we have previously dem- analytes may also hold promise as antigen-stimulated biomarkers onstrated (19), we found that immunomodulatory effects of of treatment response. Resolution of thrombocytosis is another vitamin D were observed in patients having the TT and Tt geno- well-recognized phenomenon associated with tuberculosis treat- types of the TaqI VDR polymorphism. This observation suggests ment (29), and network analysis revealed this to be linked to that if these responses can be augmented—by administering vi- a decrease in circulating CCL5 and antigen-stimulated CCL5 and tamin D at higher doses, for example—then tuberculosis patients IL-4 among patients in our study. Although best known for their might derive a clinical benefit from vitamin D supplementation role in hemostasis, platelets are also recognized to secrete CCL5 irrespective of TaqI genotype. More broadly, the ability of vitamin (30), which can enhance production of IL-4 by CD4+ T cells (31).
D to accelerate resolution of potentially immunopathological in- The role of platelets in the antimycobacterial response warrants flammatory responses without compromising bacterial killing rai- ses the possibility that supplementation might also have benefits in Having characterized the immune response to antituberculous patients receiving antimicrobial therapy for pneumonia and sepsis, therapy, we proceeded to investigate how this was affected by in whom failure to resolve hypercytokinaemia is associated with administration of adjunctive vitamin D. In contrast to studies increased mortality (5, 6).
investigating immunomodulatory actions of vitamin D supple-mentation in healthy people and in those with noncommunicable Materials and Methods diseases (32–35), we report pleiotropic immunomodulatory Details of the trial protocol have previously been reported; participants were actions of vitamin D in tuberculosis patients. This difference may randomized to receive four fortnightly doses of 2.5 mg vitamin D3 vs. placebo reflect the very high prevalence of profound deficiency at base- in addition to standard antituberculous therapy (19). Antigen-stimulated line among participants in our study; the relatively high dose of whole-blood assays were performed as previously described (42). Concen- vitamin D administered; or the fact that MTB can up-regulate trations of IL-1β, IL-1RA, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12 (p40/p70), expression of the vitamin D 1-α hydroxylase CYP27B1 to gen- IL-13, IL-15, IL-17, G-CSF, GM-CSF, IFN-α, IFN-γ, TNF, CXCL8, CXCL9, CXCL10, erate immunomodulatory concentrations of calcitriol at sites of CCL2, CCL3, CCL4, CCL5, CCL11, EGF, FGF-β, HGF, and VEGF were quantified infection (11). Among patients fulfilling criteria for per-protocol using a human 30-plex bead immunoassay panel (Invitrogen). Serum CRP analysis (n = 96), vitamin D accelerated sputum smear conver- and albumin concentrations were assayed using an Architect ci8200 analyzer(Abbott Diagnostics). Serum PGE2 concentration was analyzed by high-sensi- sion (P = 0.04). This finding contrasts with results of our pre- tivity competitive enzyme immunoassay (Assay Designs). Concentrations of viously published intention-to-treat analysis (n = 126), in which LL-37, HNP1-3, and NGAL were analyzed by ELISA (Hycult Biotechnology).
a trend toward faster conversion in vitamin D-supplemented Concentrations of MMP-1, -2, -3, -7, and -8 were determined by Fluorokine patients did not attain statistical significance (19). This differ- MAP multianlalyte profiling (R&D Systems); concentration of MMP-9 was ence may reflect the superior compliance of participants in- determined by DuoSet ELISA (R&D Systems). Antigen-stimulated AMP and cluded in the per-protocol analysis, which excluded patients who MMP concentrations were corrected by subtraction of unstimulated values.
did not take a full course of study medication. Vitamin D also Full blood counts were performed using a LH750 hematology analyzer suppressed circulating concentrations of IFN-γ and IFN-γ-in- (Beckman Coulter). ESR was measured by the Wintrobe method using ducible chemokines CXCL9 and CXCL10, MMP-9, and antigen- a s2000 analyzer (Desaga). DNA extraction and genotyping were performed stimulated Th1 responses. These in vivo findings are consistent as previously described (19).
with reported immunomodulatory actions of calcitriol in vitro (8, PCA was conducted using Qlucore Omics Explorer 2.2 (Qlucore). Analyte 36, 37). In contrast to these suppressive actions, vitamin D also concentrations were log2-converted and normalized to the mean for each attenuated treatment-induced falls in antigen-stimulated CCL5, analyte with variance −1 to +1. Rank-regression analysis was applied to PCA- IL-4, and IFN-α. IL-4 has recently been reported to induce ex- transformed data to identify parameters whose concentration was affected by pression of CYP24A, the principal catabolic enzyme of both antituberculous therapy (by making within-patient comparison of samples at calcidiol and calcitriol (38); the increase in antigen-stimulated different time points among patients allocated to placebo) and vitamin D (by IL-4 secretion observed in the intervention arm of the study may making between-patient comparison of samples from patients allocated to therefore represent part of a negative-feedback loop via which placebo vs. vitamin D at each time point). This analysis yielded t statistics calcitriol regulates its own concentration at the site of disease.
(calculated as the regression coefficient for each parameter divided by its SD)representing the magnitude of difference in concentration of a given pa- The finding that administration of vitamin D enhanced antigen- rameter between groups being compared; P values, representing the proba- stimulated IFN-α responses is of particular interest, given the bility that such differences could have arisen by chance alone; and q values, pivotal role of type 1 interferons in antiviral responses (39), and representing the lowest false discovery rate for which differences would be the clinical observation of a sixfold reduction in upper re- accepted as statistically significant under the Benjamini–Hochberg procedure spiratory tract infections among patients in the intervention arm for multiple-testing correction (43). The effects of allocation on circulating of the trial (19). Modulation of antigen-stimulated responses by immune responses at 8 wk within genetically defined subgroups were ana- vitamin D supplementation may represent changes in numbers of lyzed using Mann–Whitney U tests. The effect of allocation on time to sputum circulating lymphocyte subpopulations or direct effects of vita- clearance was analyzed by Cox regression analysis, adjusting for age, ethnicity, min D on lymphocyte function. More detailed characterization baseline sputum smear, neutrophil count, and presence or absence of cavita- of the effects of vitamin D supplementation on numbers and tion on baseline chest radiograph as previously described (19).
cytokine profiles of lymphocyte subsets is warranted.
Further details are presented in .
Coussens et al.
PNAS Early Edition 5 of 6 ACKNOWLEDGMENTS. We thank Dr. Delmiro Fernandez-Reyes (Medical acting as Principal Investigators; the members of our Data Monitoring Research Council National Institute of Medical Research) for advice on Committee, Dr. Guy E. Thwaites (Chair), Dr. Brenda E. Jones, and Dr. Tuan statistical analysis; Dr. Alleyna Claxton (Homerton University National Q. Phung; and all the tuberculosis nurses and administrative staff who Health Service Foundation Trust) for assistance with development of referred patients to the study and all patients who participated in the microbiological outcome measures; Drs. John C. Moore-Gillon (Barts and trial. The study was funded by the British Lung Foundation (TB05/11) and The London National Health Service Trust), Thomas C. Stokes (Queen the United Kingdom Medical Research Council (U1175 22141). Merck Elizabeth Hospital), and Stefan Lozewicz (North Middlesex Hospital) for Serono donated study medication.
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Supporting Information Coussens et al. 10.1073/pnas.1200072109 SI Materials and Methods IL-1β, IL-1RA, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12 Participants. The patients included in this study were participants in (p40/p70), IL-13, IL-15, IL-17, G-CSF, GM-CSF, IFN-α, IFN-γ, the AdjuVIT study: a double-blind randomized placebo-controlled TNF, CXC chemokine ligand 8 (CXCL8), CXCL9, CXCL10, trial of high-dose vitamin D during intensive-phase antimicrobial chemokine ligand 2 (CCL2), CCL3, CCL4, CCL5, CCL11, EGF, treatment of pulmonary tuberculosis (TB), conducted in London, FGF-β, hepatocyte growth factor (HGF), and VEGF were United Kingdom. A detailed account of study design has previously quantified using a human 30-plex bead immunoassay panel been given (1). Briefly, 146 adults with smear-positive pulmonary (sensitivity according to Lot #617361; Invitrogen). Serum sam- TB were randomized to receive four doses of 2.5 mg vitamin D ples required high dilution for accurate determination of CCL5 placebo in addition to standard intensive-phase antituberculous concentration and all were reassayed using a single-plex bead therapy. The first dose of study preparation was administered assay (Invitrogen). Serum PGE2 concentration was analyzed by within 7 d of the start of antituberculous therapy, and subsequent high sensitivity competitive enzyme immunoassay (EIA; Assay doses were administered 14, 28, and 42 d after the start of treat- Designs; sensitivity 13.4 pg/mL). Plasma concentrations of cath- ment. Baseline assessment included collection of a sputum sample elicidin (LL-37; sensitivity 31 pg/mL), human neutrophil peptides for microscopy and culture and a blood sample. Fresh whole blood (HNP1-3; sensitivity 156 pg/mL) and neutrophil gelatinase-asso- was sent for determination of full-blood count and erythrocyte ciated lipocalin (NGAL; sensitivity 400 pg/mL) were analyzed by sedimentation rate (ESR) and ex vivo stimulation with mycobac- ELISA (Hycult Biotechnology). Plasma concentrations of MMP- terial antigens as described below. Aliquots of serum, plasma, and 1, -2, -3, -7, and -8 were determine by Fluorokine Multianlalyte whole blood were also stored at −80 °C until completion of the Profiling (sensitivity according to Lot #273379; R&D Systems); trial. Participants were reviewed at 14, 28, 42, and 56 d after starting plasma concentration of MMP-9 was determined by DuoSet antituberculous therapy to assess clinical status and to monitor for ELISA (sensitivity 3 pg/mL; R&D Systems). Multiplex bead as- adverse events. Blood and sputum samples were collected at each says were performed on a Luminex 200 anlayzer (Luminex).
time point and processed as above. Full characterization of the ELISA and EIA absorbances were measured using a Benchmark immune response to antituberculous therapy and adjunctive vita- Plus microplate spectrophotometer (Bio-Rad Laboratories). The min D was performed in the subset of participants who fulfilled concentrations of 39 of these analytes (all of the above except predefined criteria for per-protocol analysis (i.e., those infected PGE2, CRP, and albumin) were also determined in supernatants with a rifampicin-sensitive isolate of Mycobacterium tuberculosis of rESAT-6– and rCFP-10–stimulated whole blood, as above; who received at least three doses of study preparation, who were preliminary determination of IFN-γ concentration in supernatants compliant with antituberculous therapy, who did not take second- of baseline whole blood samples stimulated with the six-antigen line antituberculous therapy or oral corticosteroids, who com- panel was performed using ELISA (Cellestis). Antigen-stimulated pleted all study visits and who were not HIV sero-positive). The AMP and MMP concentrations were corrected by subtraction of study was approved by East London and The City Research Ethics unstimulated values. For MMP-2, -3, -8, and HNP and NGAL, Committee (06/Q0605/83), and registered with unstimulated values were generally greater than stimulated values, (NCT00419068). Written informed consent was obtained from all and this was the case sometimes for MMP-9 and LL-37. Cytokine/ participants before enrolment.
chemokine values were generally undetectable in unstimulatedsamples and a correction was not applied.
Antigen-Stimulated Whole-Blood Assay. Fresh whole blood was di- Fourteen hematological parameters [lymphocyte, monocyte, luted 1:10 in RPMI medium 1640 (Sigma-Aldrich) and duplicate neutrophil, eosinophil, basophil, RBC and platelet counts, he- 180-μL aliquots were stimulated in 96-well plates at 37 °C in the moglobin concentration (Hb), mean corpuscular hemoglobin presence of 5% CO2 with the following: recombinant early secreted (MCH), MCH concentration (MCHC), mean cell volume antigenic target 6 kDa, (rESAT-6) (Rv3875; Proteix Biotechnolo- (MCV), packed cell volume (PCV), red cell distribution width gies; final concentration 2.5 μg/mL), recombinant culture filtrate (RDW), and ESR] were also measured in fresh whole blood.
protein 10 kDa (rCFP-10) (Rv3874; Proteix Biotechnologies; final Full blood counts were performed using a LH750 hematology concentration 2.5 μg/mL), peptide pools (15-mers overlapping by analyzer (Beckman Coulter). ESR was measured by the Wint- 10 residues) for ESAT-6, CFP-10, and TB7.7 (Rv2654; all from robe method using an s2000 analyzer (Desaga).
Peptide and Protein Research; final concentration 5 μg/mL), tu-berculin-purified protein derivative (Statens Serum Institute; final DNA Extraction and Genotyping. DNA was extracted from whole concentration 20 μg/mL) and 2% (wt/vol) BSA in PBS (negative blood using the Promega Wizard SV 96 Genomic DNA Purifi- control). Plates were centrifuged after 72-h incubation, and cell- cation System on the Biomek FX robot (Beckman Coulter), free supernatants were aspirated and frozen at −80 °C pending quantified using the Nanodrop spectrophotometer and normal- immunological analysis. Recombinant antigens were tested for ized to 5 ng/mL per 10 ng DNA was used as template for a 5 mL presence of endotoxin: concentrations were found to be 1,723 IU predeveloped TaqMan assay (Applied Biosystems) to type the (EU)/mg for rESAT-6 and 260 IU (EU)/mg for rCFP-10. Control TaqI polymorphism (rs731236) of the vitamin D receptor. This experiments to determine the effects of adding these concen- assay was performed on the ABI 7900HT platform in 384-well trations of endotoxin to whole blood were not performed.
format, and data were analyzed with Autocaller software.
Immunological Analysis. Concentrations of 42 soluble factors [C Statistical Analysis. Analyte concentrations were calculated from reactive protein (CRP), albumin, and a panel of 18 cytokines, 8 raw ELISA data using Masterplex 2010 software (Hitachi Solutions chemokines, 4 angiogenic factors, 3 antimicrobial peptides America) and GraphPad Prism 5 software. Principal component (AMP), 6 matrix metalloproteinases (MMP), and prostaglandin analysis (PCA) was conducted using Qlucore Omics Explorer 2.2 E2 (PGE2)] were determined in serum/plasma as follows. Serum (Qlucore). Analyte concentrations were log2-converted and nor- CRP and albumin concentrations were assayed using an Architect malized to the mean for each analyte with variance −1 to +1.
ci8200 analyzer (Abbott Diagnostics). Serum concentrations of Missing values were imputed by K nearest neighbors (2); they arose when a sample was not taken at a particular time point, or nicity, and age, were adjusted for by using the eliminated factors when a sample from a particular time point was of inadequate approach applied to the sample annotation "patient ID," which volume to allow determination of the concentration of a given generates a general linear model to normalize baseline differences analyte. For circulating parameters, 2.5% of data points were between patients (3). Analytes significantly affected by the addi- missing. For ESAT-6–stimulated parameters, 4% of datapoints tion of vitamin D to antituberculous therapy were identified by were missing. For CFP-10–stimulated parameters, 3% of data- creating an interaction variable between week of treatment (weeks points were missing. The degree of sample clustering in PCA plots 0, 2, 4, 6, 8) and allocation (vitamin D vs. placebo). The 10 in- was quantified by calculating the sum of the Euclidean distances teraction groups were analyzed by rank regression (ordered by between each point in the PCA plot and all other points in the plot week 0–8 in alternating groups, placebo vs. vitamin D), eliminating at the same time point: high values of the sum of Euclidean dis- factors "week" and "patient ID" and selecting P value <0.05 and q tances represent a lower degree of "cluster tightness" at a given value <0.1. PCA networks were created using one connection, i.e., time point, and vice versa. Median values of the sum of Euclidean by connecting each analyte to one other analyte with which it distances were compared between time points using the Kruskal– shared the most similar pattern of change over time. The distance Wallis test with Dunn's post hoc analysis.
between analytes in the network represents their Pearson corre- Parameters whose concentration changed significantly during lation coefficients. The effects of allocation on circulating immune intensive phase antituberculous therapy were identified by responses at 8 wk within genetically defined subgroups were ana- analysis of samples from patients randomized to the placebo lyzed using Mann–Whitney U tests. The effect of allocation on control arm of the study, conducting a rank regression on week of time to sputum clearance was analyzed by Cox regression analysis, treatment with thresholds of P = 0.05 for statistical significance adjusting for age, ethnicity, baseline sputum smear, neutrophil and q = 0.1 for false discovery rate. Differences in baseline count, and presence or absence of cavitation on baseline chest characteristics between patients, such as extent of disease, eth- radiograph, as previously described (1).
1. Martineau AR, et al. (2011) High-dose vitamin D(3) during intensive-phase antimicrobial 3. Wichura MJ (2006) The Coordinate-Free Approach to Linear Models (Cambridge Univ treatment of pulmonary tuberculosis: A double-blind randomised controlled trial. Lancet Press, Cambridge).
2. Troyanskaya O, et al. (2001) Missing value estimation methods for DNA microarrays.
464 assessed for eligibility 214 ineligible for randomisation104 declined to participate 146 enrolled and randomised 73 allocated to vitamin D 73 allocated to placebo 29 excluded from per-protocol 22 excluded from per-protocol - 2 sputum culture negative - 1 sputum culture negative - 6 isolate other than M. tuberculosis - 3 isolate other than M. tuberculosis - 0 with rifampicin-resistant isolate - 4 with rifampicin-resistant isolate - 10 received < 3 doses of study - 6 received < 3 doses of study - 3 diagnosed with HIV infection - 2 diagnosed with HIV infection - 5 took oral corticosteroids - 3 took oral corticosteroids - 2 interrupted treatment - 1 interrupted treatment - 1 non-compliant with treatment - 2 non-compliant with treatment - 2 took second-line therapy - 3 took second-line therapy 44 included in analysis of circulating 51 included in analysis of circulating 25 – antigen-stimulated whole blood 23 – antigen-stimulated whole blood assays not performed assays not performed 19 included in analysis of antigen- 28 included in analysis of antigen- stimulated responses stimulated responses Trial profile. We assessed 464 patients for eligibility to participate in the trial between January 25, 2007 and July 3, 2009; 214 were ineligible, 104 were eligible but declined randomization, and 146 were randomized. Of these subjects, 95 patients (44 allocated to vitamin D, 51 allocated to placebo) fulfilledpredefined criteria for per-protocol analysis and contributed samples to the analysis of circulating immunological parameters. Antigen-stimulated whole-bloodassays were performed on samples taken from all study participants who were recruited on or after May 15, 2008; 47 of these participants (19 allocated tovitamin D, 28 allocated to placebo) fulfilled per-protocol criteria and contributed samples to the analysis of antigen-stimulated responses. †Some participantswere ineligible to enter per-protocol analysis for more than one reason.
Sum of Euclidean distances for principal component analysis plots presented in Median values of the sums of the Euclidean distances between each point in the PCA plot and all other points in the plot at the same time point are compared between time points using the Kruskal–Wallis testwith Dunn's post hoc analysis: high values of the sum of Euclidean distances represent a lower degree of cluster tightness at a given time point, and vice versa.
(A) Circulating immunological parameters: baseline samples were less tightly clustered than follow-up samples (median sum of Euclidean distances at 0 vs. 4 vs.
8 wk, 253.5 vs. 197.1 vs. 203.4, respectively; P < 0.0001). (B) rESAT-6–stimulated responses: samples converged from a loosely clustered pattern at baselinetoward a more tightly clustered pattern at 4 wk, and then back to a more loosely clustered pattern at 8 wk (median sum of Euclidean distances at 0 vs. 4 vs.
8 wk, 85.4 vs. 66.7 vs. 83.3, respectively; P < 0.0001). (C) rCFP-10–stimulated responses: pattern as for rESAT-stimulated responses (median sum of Euclideandistances at 0 vs. 4 vs. 8 wk, 91.9 vs. 67.0 vs. 89.3, respectively; P < 0.0001). Lines at median. ***P < 0.0001. NS, P > 0.05.

Concentrations of IFN-γ in supernatants of antigen-stimulated whole blood. Blood was stimulated for 72 h with the following antigens: tuberculin- purified protein derivative (PPD), peptide (pep) TB7.7, recombinant (r) and peptide ESAT-6 and CFP-10. Assays were performed on blood samples of 47 patientssampled within 1 wk of initiating antituberculous therapy before administration of adjunctive vitamin D or placebo. Horizontal bar at median. 1 IU/mL IFN-γapproximates to 40 pg/mL IFN-γ.
Antigen-stimulated IFN-γ secretion is not influenced by administration of antimicrobials or adjunctive vitamin D during intensive phase antituber- culous therapy. Data from 47 patients (28 allocated to adjunctive placebo, dotted line, and 19 allocated to adjunctive vitamin D, solid line) sampled at 0, 2, 4, 6,and 8 wk into therapy are presented. (A) Total rESAT-6–stimulated IFN-γ secretion by allocation. (B) Total rCFP-10–stimulated IFN-γ secretion by allocation. (C)rESAT-6–stimulated IFN-γ secretion per 106 lymphocytes by allocation. (D) rCFP-10–stimulated IFN-γ secretion per 106 lymphocytes by allocation. Medians andinterquartile (IRQ) ranges are presented.

Three-dimensional network PCA plots showing the effect of adjunctive vitamin D supplementation on the immune response to intensive-phase antituberculous therapy. Individual points represent a single analyte. The distance between analytes is a measure of their Pearson correlation coefficients. Lineslink analytes to their nearest neighbor, the analyte with the most similar pattern of change during the course of treatment. Analytes are colored by R2-statistic:blue represents suppression and yellow augmentation by vitamin D. Component vectors are displayed, along with a percent figure signifying the proportion ofthe variability in the data that each component accounts for. (A) Circulating immune parameters. (B) rESAT-6–stimulated whole-blood responses. (C) rCFP-10–stimulated whole-blood responses.
Baseline characteristics of participants entering analyses of circulating and antigen-stimulated responses by allocation Circulating responses (n = 95) Antigen-stimulated responses (n = 47) Vitamin D (n = 44) Vitamin D (n = 19) Median age, years (IQR) 31.8 (24.4–41.8) 30.1 (24.7–36.6) 29.4 (23.7–37.5) 31.0 (23.9–36.6) Asian/Asian British Black/Black British White/Latin American Median symptom duration prediagnosis, Median duration of treatment pre-enrolment, Baseline sputum smear ≤3 AFB per high-power field >3 AFB per high-power field Serum 25(OH)D, nmol/L Serum 25(OH)D <20 nmol/L Serum 25(OH)D <75 nmol/L Body mass index, kg/m2 Baseline chest radiograph TaqI vitamin D receptor genotype Data are number (%) or mean (SD) except where stated. AFB, acid-fast bacilli; IQR, interquartile range; 25(OH)D, 25-hydroxyvitamin D.
Influence of intensive-phase antituberculous therapy on circulating and antigen-stimulated immune responses in adults with smear-positive pulmonary tuberculosis Circulating responses (n = 51) responses (n = 28) responses (n = 28) Factors and parameters Angiogenic factors Antimicrobial peptides Acute phase proteins Red blood cell parameters White blood cell parameters Neutrophil count{ Lymphocyte count{ Eosinophil count{ MCV, mean cell volume; PCV, packed cell volume; RDW, red cell distribution width; —, no data.
*t-Statistic (regression coefficient/SD) represents magnitude of change over time of treatment; sign indicates direction of this change. t-statistic value notpresented when P > 0.05.
†P values derived using principal component analysis and rank regression on treatment duration; all q values were < 0.1.
‡Median baseline concentration below limit of detection in serum samples.
§Median baseline concentration below limit of detection in whole blood assay supernatants.
{Not determined in whole blood assay supernatants.
Influence of adjunctive vitamin D supplementation on circulating and antigen-stimulated immune responses to intensive-phase antituberculous therapy Circulating responses responses (n = 47) responses (n = 47) Factors and parameters Angiogenic factor Antimicrobial peptides Matrix Metalloproteinase Acute phase protein Red blood cell parameter White blood cell parameters Lymphocyte count§ RDW, red cell distribution width; —, no data.
*t-statistic (regression coefficient/SD) represents magnitude of difference between patients receiving adjunctive vitamin D vs. placebo;sign indicates direction of this difference for vitamin D vs. placebo arms. t-statistic value not presented when P > 0.05.
†P values derived using principal component analysis and rank regression on the interaction term "treatment duration*allocation"; all qvalues were < 0.1.
‡Median baseline concentration below limit of detection in serum samples.
§Not determined in whole blood assay supernatants.

Three dimensional principal component analysis plots generated using parameters that significantly contribute to the effect of intensive-phase antituberculous therapy on the immune response in patients with smear-positive pulmonary tuberculosis. Individual points represent one patient at one timepoint and their position in the plot is determined by the combined effects of all parameters measured for that patient sample that contribute significantly tothe effect of antituberculous therapy. The distance between sample points presents Euclidean distance and is calculated using all parameters which contributesignificantly to the effect of antituberculous therapy. Component vectors are displayed, along with a percent figure signifying the proportion of the variabilityin the data that each component accounts for. Color gradient denotes timing of sample, with baseline samples in red and 8-wk samples in yellow. (A) Cir-culating immunological parameters; (B) rESAT-6–stimulated responses; (C) rCFP-10–stimulated responses.

Three dimensional network principal component analysis plots showing the effect of intensive-phase antituberculous therapy on the immune response in patients with smear-positive pulmonary tuberculosis. Individual points represent a single analyte. The distance between analytes is a measure oftheir Pearson correlation coefficients. Lines link analytes to their nearest neighbor, the analyte which has the most similar pattern of change during the courseof treatment. Analytes are colored by R2-statistic: blue represents a decrease, and yellow an increase, with time of treatment. Component vectors are displayed,along with a percent figure signifying the proportion of the variability in the data for which each component accounts. (A) Circulating immune parameters; (B)rESAT-6–stimulated responses; (C) rCFP-10–stimulated responses.

Three dimensional principal component analysis plots generated using parameters that significantly contribute to the effect of adjunctive vitamin D on the immune response to intensive-phase antituberculous therapy. Individual points represent one patient at one time point and their position in the plotis determined by the combined effects of all parameters measured for that patient sample that contribute significantly to the effects of adjunctive vitamin D;data from all five time points are displayed. The distance between sample points presents Euclidean distance and is calculated using all parameters whichcontribute significantly to the effects of adjunctive vitamin D. Component vectors are displayed, along with a percent figure signifying the proportion of thevariability in the data for which each component accounts. (A) Circulating immunological parameters in patients randomized to vitamin D (orange, n = 44) vs.
placebo (gray, n = 51). (B) rESAT-6–stimulated responses in patients randomized to vitamin D (orange, n = 19) vs. placebo (gray, n = 28). (C) rCFP-10–stimulatedresponses in patients randomized to vitamin D (orange, n = 19) vs. placebo (gray, n = 28).

Source: http://www.theoldexchangesurgery.co.uk/website/D81623/files/Vitamin_D_info.pdf