Are the Immunomodulatory Properties of Vitamin D Dependent Upon Route of Supplementation? Catherine Hawrylowicz Kylie Morgan, Antony Young Vitamin D & human health Vitamin D insufficiency is a worldwide epidemic (>1 billion affected) Vitamin D insufficiency correlates with immune disease presentation & severity • Autoimmune: IBS, RA, MS • Respiratory: Asthma, Allergy, COPD Bacterial & viral infections Low UVR exposure gradient effect • Multiple sclerosis, Type 1 diabetes • Peanut allergy in Australia Holick M, 2007. NEJM; Morgan K et al, 2017. Photochem Photobiol Sci ttp://www.australianallergycentre.com.au/wp-content/uploads/2015/09/australia-peanut-allergy.png Systematic reviews & meta-analyses of individual participant data support vitamin D supplementation Joliffe DA et al, 2017. Lancet Respir Med 5(11):881-890. Vitamin D supplementation reduced the rate of asthma exacerbations requiring treatment with systemic corticosteroids overall…….. Martineau AR et al, 2017 BMJ 356:i6583 Vitamin D supplementation was safe, protected against acute respiratory tract infection overall; greatest in very vitamin D deficient subjects when given daily/weekly; not bolus Immunological mechanisms underpinning associations of vitamin D with respiratory health i. Effective clearance of pathogens ➢Vitamin D enhances multiple antimicrobial mechanisms e.g. cathelicidin, beta-defensins, autophagy J Adams, M Hewison, J White & others ii. Controlled inflammation ➢Vitamin D enhances multiple immune regulatory/anti inflammatory pathways iii. Enhanced clinical & anti-inflammatory response to glucocorticoids ➢ Vitamin D favourably modifies IL-10 / IL-17A ratios Pfeffer & Hawrylowicz, 2018. CHEST IMMUNI-D study: Are the Immunomodulatory Properties of Vitamin D Dependent Upon Route of Supplementation? • Detrimental impact UVR exposure: Erythema, Photoageing, DNA damage-ROS, Photocarcinogenesis/skin cancer • Immunological differences: Reduced responses to vaccination; Modulation via additional non-vitamin D mediated pathways VDR knockout mice equally susceptible to UVR-induced immune suppression IMMUNI-D study: Are the Immunomodulatory Properties of Vitamin D Dependent Upon Route of Supplementation? • Design protocols that comparably increase vitamin D status through SSR and Vitamin D supplementation • Compare immune changes after restoration of vitamin D sufficiency by UVR versus supplementation in a vitamin D deficient healthy population by assessing changes in: • Peripheral immune populations • Dendritic cell maturation and activation • Global changes to dendritic cell gene expression IMMUNI-D study: Restoration of vitamin D sufficiency increases frequency Foxp3+ Treg YOUNG HEALTHY ADULTS, vitamin D insufficient (<50nmol), Fitzpatrick skin type I & II; n=8 or 9/group 3 interventions: None (control) UV Radiation (1.25 SED SSR twice weekly; total 10) 4000 IU/day cholcalciferol (vitamin D3) for 28d Supplementation UV Radiation Control IMMUNI-D study: Restoration of vitamin D sufficiency increases frequency Foxp3+ Treg YOUNG HEALTHY ADULTS, vitamin D insufficient (<50nmol), Fitzpatrick skin type I & II 3 interventions None (control) UV Radiation (1.25 SED SSR twice weekly; total 10) 4000 IU/day cholcalciferol (vitamin D3) for 28d Large flow cytometry panel of major PBMC populations; Assessed d0 vs. d28 No difference in lymphocytes (B cells, CD4, CD8, NK, NKT, monocyte% Fox Psubsets)3 s 15 e R = 0.5491 t p=0.035 p=0.014 y c P <0.001 o h p m y 10 L + 3 D C n i 5 3 P x o F % 0 0 20 40 60 80 100 25(OH)D nmol/L IMMUNI-D DC findings 1. Dendritic cell maturation and activation: Vitamin D promotes a tolerogenic phenotype - IMMUNI-D: little or no impact on mDC or pDC frequency or maturation in the periphery in the steady state - mDC activation reduced in culture when vitamin D levels were higher e.g. HLA-DR, CD86, CD83 mDC 2. Global changes to dendritic cell gene expression - Myeloid DC (mDC) & plasmacytoid DC (pDC) - Differentially expressed genes determined by Microarray - Pathway analysis performed (IPA) pDC 1.5 fold change, p</= 0.005 threshold UVR and vitamin D supplementation increase gene expression of the vitamin D receptor & the antimicrobial peptide cathelicidin in mDC VDR CD83 p=0.004 5 20 1015 p=0.037 5 4 e e g g 4 n n 3 a a h h c 3 c d d l l 2 o o 2 F F 1 1 VDR, vitamin D receptor 0 0 CAMP, cathelicidin CTL UVR SUP CTL UVR SUP CD83, DC maturation marker CYP27B1 CAMP 5 p=0.079 4 4 p=0.0043 e g 3 n a 3 h e g C 2 n d a l h o C 2 1 F d l o 0 F 1 -1 CTL UVR SUP 0 CTL UVR SUP Differentially expressed genes mDC Vitamin D SupplementationVolcano Plot of 1/ANOVA-3way:2 1*10 -4 C3orf19 ) 0 y a 1*10 -3 D * n o i t a t n IFIT1 e m e IFIT3 l p p u S OAS2 * HSD17B4 d i o l e y LOC728942 MYD88 m . NAGA s -2 v 1*10 TINF2 8 2 y ANKRD54 a IFIT2 D * n OAS1 EPSTI1 o i RNU6-15 t a t SETDB2 RBM14 SAMD9 n ISG15 e m RING1 MORG1 SLAMF8 e l p ZNF845 C13orf15 CTNNA1 TMCO1 SLC2A4RG p u PIK3AP1 DCAF7 S ASB1 LOC144438 GCA SCARB2 * PAK1 d i LOC728519 o ELK1 l TAF13 SLC20A1 e -1 NDUFB5 MX1 YARS y 1*10 GIMAP4 NAGPA RRM1 m SNORD13 ( MAN2B2 FRAT2 e IFI6 u ATP6V1B2 l ZNF581 MEF2D SHMT2 a DNMT1 v ZSWIM7 - DEXI p PAQR7 CYTH1 ECGF1 RNU4-1 GMEB2 PLAC8 CEBPD NFE2 SNORD48 LOC731682 SNORD46 1 -4 -2 N/C 2 4 Fold-Change(myeloid * Supplementation * Day 28 vs. myeloid * Supplementation * Day 0) Myeloid DC – pathway analyses Control Supplementation UVR pg/ml cytokine Future directions Immune effects of UVR vs. supplementation - Bioinformatic analysis of gene transcription studies of mDC and pDC pre/post UVR vs. vitamin D supplementation ongoing - Biopsies from unexposed skin in a subset of IMMUNI-D participants available to study Treg in tissues PM effects on immune function - Use of real world PM samples (LEZ) - DC, T cells, neutrophils - Identification of signaling pathways (AhR, Calcium, endotoxin, oxidative stress etc) - Effects vitamin D comparably with different PM samples, signalling pathways – clinical effects may be stronger in children than adults; pregnancy data Further information IMMUNI-D (UVR vs. Supplementation) Study Kylie Morgan, Antony Young Vitamin D and particulate matter air pollution studies Beth Mann, Paul Pfeffer, Tzer-Ren Ho, Drew Glencross Ian Mudway 1. Characterisation of real world air pollution extracts from London e.g. Oxidative Stress, Particulate matter size, Metal Composition, Endotoxin, Organic fraction of PM 2. Comparison of real world air pollution extracts from London with well-established standards and controls in influencing the phenotype and function of primary peripheral human DC and macrophages; also HBECs. 3. How do PM-stimulated DCs affect the adaptive immune system (CD4 & CD8 T cells)? Matthews N et al, 2014 & 2016. Am J Resp Cell Mol Biol 3. What component(s) of pollution extracts modulate immune? 4. Can vitamin D mitigate the detrimental impact of pollution extracts on immune function? Mann EH et al, 2017. Am J Resp Cell Mol Biol IMMUNI-D study population • Healthy (non allergic/asthmatic, no immunomodulating or photosensitising medications) • Young population (18-40yrs old) • No previous sun exposure in the previous 3 months • No vitamin D supplementation in the previous 3 months • Fitzpatrick skin type (I/II) Treatmen Gender Mean Skin Type BSA (m2) BMI Mean VD3 t group M F Age (± I II (± SD) (± SD) (nmol/L) SD) (± SD) Control 5 8 25 0 13 1.76 21.7 29.73 (10.18) (4.3) (0.16) (2.21) UVR 5 8 25 0 13 1.77 22.3 36.31 (9.13) (5.1) (0.16) (2.04) Supp. 4 8 28 2 11 1.73 22.9 29.13(10.61) (3.9) (0.15) (2.67) Phase 1 (2015/2016): Phase II (2016/2017): n=4 per group (n=12 total) n= 8/9 per group (n= 27 total) Detrimental Impact of UVR exposure • Erythema • Photoaging • DNA damage -> ROS • Photocarcinogenesis/skin cancer • Current health guidelines recommend limiting UVR exposure - contributing factor to vitamin D deficiency • Risks vs. benefits of UVR exposure? • Immunological differences: • Vaccination - reduced responsiveness • Immunomodulation via additional non-vitamin D mediated pathways • VDR knockout mice equally susceptible to UVR-induced immunesuppression Vitamin D and asthma IMMUNI-D study No significant changes in major immune cell population frequency % Lymphocytes CD14+ monocytes NKT cells B cells CD4+ T cells 40 Lymphocytes 100 2.5 10 80 s 30 80 2.0 8 e 60 t y s c l l NKT cells + o + + e 4 60 h 1.5 4 9 c 6 1 p 20 1 D D T 40 C D m C K y C L N 40 % % 1.0 4 % % 10 % 20 20 0.5 2 0 0 0 CTL UVR SUP 0.0 0 CTL UVR SUP CTL UVR SUP CTL UVR SUP CTL UVR SUP % Monocytes CD14+ CD16+ monocytes NK cells T cells CD8+ T cells 6 5 10 100 40 4 8 80 + s 30 6 e 4 t 1 s y Monocytes l D + c l 3 + C 6 8 o e 60 3 c n D + D o 20 C 4 K C 1 M N % D 2 4 NK cells 2 % 40 C % % % 10 1 2 20 0 0 0 0 CTL UVR SUP 0 CTL UVR SUP CTL UVR SUP CTL UVR SUP CTL UVR SUP CD16+ monocytes 15 10 + 6 1 D C % 5 0 CTL UVR SUP Control UVR Supplementation 0.0599 0.0387 0.0303 0.0428 0.0342 0.0342 0.0859 0.0731 0.0405 0.053 0.0090 n=4 1. Are the Immunomodulatory Properties of Vitamin D Dependent Upon Route of Delivery - UVB vs.