Non classical effects of vitamin D Jean-Claude Souberbielle, hôpital Necker, Paris, France

few significant dietary sources intestine UVB (290-315 nm) skin 7-dehydrocholesterol Pre-vitamin D3 intestine (extremely) few significant dietary sources Vitamin D3 (cholecalciferol) Vitamin D2 (ergocalciferol) parathyroids liver FGF23 PTH 1,25OH2D + - 25OHD 1 alpha hydroxylase 1,25OH2D Classical endocrine genomic effects intestine 24- hydroxylase + FGF23 1,25OH2D 24,25OH2D Calcitroic acid bone Serum 25OHD concentration (not 1,25OH2D!) is the marker of vitamin D status. This is a consensus!

Severe vitamin D deficiency induces rickets (osteomalacia in adults) Less severe deficiency (i.e. without any obvious mineralization defect) may favour/worsen osteoporosis and is associated with an increased fracture risk through shpt and increased risk of falls in the elderly

few significant dietary sources intestine UVB (290-315 nm) skin 7-dehydrocholesterol Pre-vitamin D3 intestine (extremely) few significant dietary sources Vitamin D3 (cholecalciferol) Vitamin D2 (ergocalciferol) liver 25OHD 1 alpha hydroxylase Non classical genomic effects 1,25OH2D DNA VDR Numerous tissues including immune cells (monocytes, macrophages, T-cells ), cardiomyocytes, endothelial cells, breast, prostate, colon (normal and tumoral), brain, and much more

few significant dietary sources intestine UVB (290-315 nm) skin 7-dehydrocholesterol Pre-vitamin D3 intestine (extremely) few significant dietary sources Vitamin D3 (cholecalciferol) Vitamin D2 (ergocalciferol) liver 25OHD FGF23 PTH 1,25OH2D + - 1 alpha hydroxylase 1,25OH2D Non genomic effects VDR-MMARS Activation tyrosine kinases Intracellular Ca++ Several tissues including muscle; β cells

«classical» effects -stimulates absorption of calcium and phosphorus by the gut - direct effects on bone -effects on kidney -control of PTH secretion Favours bone mineralisation 700-800UI /day (+calcium) reduce RR of «non vertebral» fractures in the elderly Importance of «genetic» : SNP of VDR, CYP27B1, CYP24, DBP «non classical» effects Muscle 700-1000 IU/d (+calcium) reduce RR of falls in the elderly Immune System Cardiovascular health Cancers Morbidity of pregnancy (preeclampsia, gestational diabetes..) Renoprotection All-cause mortality Others

What is the «level of evidence»? (Evidence-based Medicine) -«ecologic» studies -«observational» studies (cross-sectional, longitudinal, retrospective, prospective ). Importance of statistical «adjustments» for confounders. Don t allow to conclude to a causal relationship!! -experimental studies animal models, cell culture May explain les méchanisms of action, but often use supra-physiologic dosages. Transposition of results to human being? -interventional studies (RCTs) Meta-analyses

Consequences of mutations of the VDR gene in humans and in transgenic mice (Exon 2 or 3) Vitamin D in human health: lessons from VDR null mice. Bouillon et al Endocr Rev 2008; 29: 726-776 Alopecia Facial dysmorphy impaired intestinal calcium absorption hypocalcemic rickets growth failure hypertension with high renine cardiac hypertrophy increased sensitivity to autoimmunity and to oncogen- and chemocarcinogen-induced cancers Sensitivity to infections hypofertility Some of these anomalies are reversed by a rich calcium diet. Others are independent of calcium intake

Vitamin D and the immune system Hewison M. Vitamin D and innate and adaptative immunity Vitam Horm 2011; 86: 23-62

1-Vitamin D stimulates innate immunity Implication for the prevention of infectious diseases?

Activation of human TLR2/1 triggers a vitamin D receptor-dependent antimicrobial response Philip T. Liu* 1, Steffen Stenger* 2, Huiying Li 3, Linda Wenzel 2, Belinda H. Tan 1, Stephan Krutzik 1, Maria Teresa Ochoa 1, Jürgen Schauber 4, Kent Wu 1, Christoph Meinken 2, Manfred Wagner 5, Robert Bals 6, Andreas Steinmeyer 7, Ulrich Zügel 8, Richard L. Gallo 4, David Eisenberg 3, Martin Hewison 9, Bruce W. Hollis 10, John S. Adams 7, Barry R. Bloom 9 and Robert L. Modlin 1,11. Science 2006 ; 311 : 1770-1773) Activation of Toll-like receptor 2/1 in monocytes/macrophages by specific proteins of Mycobacterium Tuberculosis induces the transcription of the VDR and CYP27B1 genes and expression of the proteins in the cytosol and ER respectively. If the extracellular 25OHD concentration is sufficient (>70 nmol/l), 25OHD enters the cells and is locally activated into 1,25OH2D which binds to the VDR. VDR associates with RXR, and the trimeric complex activates the gene coding for cathelicidin (LL37), an antimicrobial peptide able to induce bacterial destruction.

Vitamin D and respiratory infections (including TB) Observational data Poor vitamin D status is significantly associated with an increased risk of both upper and lower respiratory tract infections as reviewed in a recent meta-analysis (4 cross-sectional, 8 case-control, and 13 cohort studies of overall good quality); This was the case in adults as well as in children Jolliffe D et al. Vitamin D in the prevention of acute respiratory infection: Systematic review of clinical studies J Steroid Biochem Mol Biol 2013; 136: 321-329

Vitamin D and respiratory infections (including TB) Intervention studies Results from RCTs are conflicting. In the most recent meta-analysis, among 14 RCTs testing the effect of vitamin D supplementation on acute respiratory infection, 7 were «positive», and 7 were «null». Among these 14 trials, 6 were done in children (4 positive, and 2 «null») Among the «null» trials, vitamin D dose was small in some (i.e : 1400 IU/week), subjects were not vitamin D deficient in others, while administration of large bolus dose in others may have impaired the immune response Jolliffe D et al. Vitamin D in the prevention of acute respiratory infection: Systematic review of clinical studies J Steroid Biochem Mol Biol 2013; 136: 321-329

Vitamin D and HIV, HBV, HCV Observational data -Metha S et al AIDS 2011; 25: 579-585. 884 HIV pregnant untreated Tanzanian women. Increased risk of clinical progression during a 70-month follow-up period and of mother-to-child HIV transmission in those with 25OHD<32 ng/ml -Viard JP et al. AIDS 2011; 25: 1305-1315 1985 patients (83% on antiretroviral treatment) from the EUROSIDA cohort. Those in the lowest 25OHD tertile (<12 ng/ml) had an increased risk of AIDS-defining events and all cause deaths after multiple adjustment. -Terrier B et al Low 25-OH vitamin D levels correlate with severe fibrosis in HIV-HCV co-infected patients with chronic hepatitis. Journal of Hepatology 2011; 55: 756-761.

Vitamin D and HIV, HBV, HCV Intervention studies Abu-Mouch S et al Vitamin D supplementation improves sustained virologic reponse in chronic hepatitis C (genotype 1)-naïve patients World J Gastroenterol 2011; 17: 5184-5190 Nimer A, Abu-Mouch S Vitamin D improves viral response in hepatite C genotype 2-3 naïve patients World J Gastroenterol 2012; 18: 800-805

2-(many) Experimental data indicate that vitamin D plays a role in adaptive immunity -VDR, 1alpha-hydroxylase are found in immune cells (dendritic cells, B, and T cells, macrophages..) constitutively or after stimulation. -1,25OH2D blocks dendritic cell maturation -1,25OH2D influences T cell gene expression of cytokines required for antigen presentation to T cells (Il1, Il2, Il12, Il17, INFgamma) -1,25OH2D inhibits the expression of inflammatory cytokines (Il6, TNF alpha) and stimulates the expression of anti-inflammatory cytokines (Il10) => Vitamin D favours Th2 and Treg phenotypes versus Th1, Th17. Role of vitamin D in (some) auto-immune diseases?

Serum 25(OH)D levels & risk of Multiple Sclerosis Prospective nested case control study >7 million US military personel All ages 257 cases of MS vs 2x controls Elevated 25(OH)D levels in whites more protective at younger ages Individuals <20 years old with 25(OH)D >40 ng/ml had 91% reduction in MS risk vs. 25(OH)D <40 ng/ml (RR=0.09, 95%CI:0.01-0.75, p=0.03) 6.1-25.3 25.4-30.1 30.2-33.9 34-39.7 39.8-61.3 Munger KL. JAMA 2006;296:2832-8.

145 MS patients; follow-up : 2.3 +/-0.6 yrs ; 25OHD measured bi-annually. Hazard ratio of relapse (adjusted for age, sex, BMI)

MS patients randomized to 20,000 IU D3/week for 1 year (n=35) versus placebo (n=33) AAR (annualized relapse rate) = 0.11 MS patients randomized To 20,000IU D3/week for 1 year (n=34) versus placebo (n=32). AAR =0,5

Vitamin D supplementation & risk of type 1 diabetes Finnish birth-cohort study (born 1966) Endpoint: type 1 diabetes by end 1997 10,366 children; 81 with type 1 diabetes Recommended supplementation during first year of life: 2,000 IU/day A daily dose of 2,000 IU was associated with a 78% reduced risk of developing type 1 diabetes compared to lower doses Hyppönen E. Lancet 2001;358:1500-3.

Giulietti A et al Vitamin D deficiency in early life accelerates type 1 diabetes in Non-obese diabetic mice. Diabetologia 2004; 47:451-462 Control NOD male D-deficient male Control NOD female D-deficient female p<0.01 p=0.05

Vitamin D and complications of type 1 diabetes Joergensen C et al. Vitamin d levels, microvascular complications, and mortality in type 1 diabetes. Diabetes Care 2011; 34: 1081-1085 227 patients DM1 median follow-up: 24 yrs 44 deaths 25OHD measured approx 3 yrs after the diagnosis, and before microalbulinuria HR for death if 25OHD <10th percentile) : 2.7 (1.1-6.7) p=0.03 No association between severe vit D deficiency and albuminuria or rétinopathy

The panel on calcium and related nutrients quickly reached consensus that serum 25OHD was the correct functional indicator of vitamin D status...hence, on this point at least, there is consensus (notably, that was not the case as recently as 5-10 years ago). Heaney R. Editorial Vitamin D : how much do we need, and how much is too much. Osteoporosis Int (2000) 11 : 553-555 1 ng/ml = 2.5 nmol/l It may be more appropriate to use health-based than population-based reference values for serum 25OHD i.e., reference limits based on avoidance of adverse health outcomes for the skeleton P Lips Endocrine Reviews (2001) 22 : 477-501.

IOM vs Endocrine Society

Two groups released recently different recommendations on the use of vitamin D. Both groups acknowledged that their recommendations were based on «musculoskeletal health» IOM (Ross AC JCEM 2011) A 25OHD serum level of 50 nmol/l (20 ng/ml) is largely sufficient and «covers the requirements of at least 97.5% of the population» Endocrine Society (Holick M JCEM 2011) Vitamin D deficiency 25OHD<50 nmol/l (20 ng/ml) Vitamin D insufficiency 25OHD = 50-75 nmol/l (20-30 ng/ml) Surprisingly, the two groups reached these different conclusions after the (extensive) analysis of virtually the same data Public health recommendations versus (individual) Patient care

Hilger J et al A systematic review of vitamin D status in populations Worldwide British Journal of Nutrition August 2013 If analysis restricted to European Children => Mean 25OHD = 50.6 nmol/l

What are the major determinants of vitamin D status? -Older age -Female gender -National policies fortification and dietary habits -Clothing practices -Skin pigmentation -Outdoor activities -BMI -Season Mithal et al, Osteoporosis Int 2009

Vitamin D intake recently recommended for children and adolescents by differents groups of experts IOM 1-18 years Ross AC et al. J Clin Endocrinol Metab 2011; 96: 53-58. EAR: 400 IU/d (10 μg) RDI: 600 IU/d (15 μg) UL : 4000 IU/d (100 μg) (3000 IU/d 1-8 yrs) Endocrine Society 1-18 years Holick M et al. J Clin Endocrinol Metab 2011; 96: 1911-1930 EAR : 600-1000 IU/d UL : 4000 IU/d Academy of Medicine (France) 0-18 years Bernard Salle et al May 2012 report EAR: 800-1000 IU/d (0-1 yr et 10-18 yrs) 600 800 IU/d (1-9 yrs) UL : 4000 IU/d Nutrition group of the French Society of Pediatrics (0-5 years and 10-18 years) Vidailhet M et al Archives de pédiatrie 2012; 19:316-328 -2 doses 100 000 IU or 80 000 IU in winter (november, february) for children 18 months-5 yrs and 10-18 yrs old -1000-1200 IU/d for breast-fed newborns (<18 months) or receiving non supplemented cow milk - 600-800 IU/d for newborns <18 months receiving vitamin D enriched milk EAR = Estimated Average Requirement RDI = Recommended Dietary Intake UL = Tolerable upper Intake level One can note that : -Mean vitamin D dietary intake in France : 92 IU/d -Official (AFSSA) EAR in France : 200 IU/d

Conclusions - Vitamin D deficiency is very frequent (in Europe mean 25OHD concentration in children is # 20 ng/ml) -Official RDI are often insufficient -Besides its classical effects on bone and mineral metabolism, vitamin D has many other potential effects on various organs. Indeed, VDR and the activating/inactivating enzyme machinery are present in almost all tissues. These «non-classical» effects are supported by many observational studies and experimental studies (animal models, cell culture ). However, results of RCTs are conflicting (positive or «null») precluding «evidence-based» recommendations. Nevertheless, all experts agree that being vitamin D deficient/insufficient is not good for health (in children and adults) and must be avoided. One remaining debate is the 25OHD level that defines vitamin D deficiency

Thank you for attention