Vitamin D: Should a regular dose be part in immunomodulation of regulatory T cells in immunomediated diseases?

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1 Vitamin D: Should a regular dose be part in immunomodulation of regulatory T cells in immunomediated diseases? Gerlies Treiber, Barbara Prietl, Thomas Pieber Department for Endocrinology and Metabolism Medical University Graz 3rd Immunology Summit, Baltimore, 3. September 214

2 The role of regulatory T-cells in autoimmunity Buckner, Nat Rev Immunol, 21

3 EffectsofVitamin D in theimmune system Treg Bouillon, Endocrine Reviews, 28

4 Vitamin D supplementationand regulatoryt cellsin healthyhumans regulatory T cells %CD4 + CD25 hi Foxp3 + CD D127 dim BL <.1 4 weeks <.1 8 weeks Pilot study 1x 14. IU n 5 Age (yrs) 31±8 Females(%) 64 BMI (kg/m²) 23.3±4.3 PrietlB, Pilz S et al IMAJ 21

5 14. IU Vit D3 Baseline r=.339 p=.9 PrietlB, Treiber G et al EurJ Nutr214

6 <.1 <.1.18 r=.64 p<.1 Significant increase in % Tregin CD4 pos T cells Unchanged Tregsuppression function in healthy humans Other immune cells not effected No treatment related side effects PrietlB, Treiber G et al EurJ Nutr214

7 RegulatoryT cellsin Typ 1 Diabetes T1D: no global deficiency in Treg cell numbers, but functional capacitylike suppressionoftregcellsisimpaired. (reviewedin Bruckner NatImmun 21) Targeting the Tregpopulation directly in vivo to increase frequency and/or function of Treg Analog of active form of vitamin D (1,25(OH) D ) increased Analog of active form of vitamin D (1,25(OH) 2 D 3 ) increased CD4 + CD25 + Treg in pancreatic lymph nodes in NOD mice. (Gregori 22) VitD3 increasedcd4 + CD25 + FoxP3 + T cellsin pancreaticlymph nodes and reduced diabetes development in NOD mice. (Takiishi Diabetes 214) 2 IU VitD3 increasedregulatorycd4 + CD25 + FoxP3 + T cells and slower decline of residual ß-cell function in new onset T1D. (Gabbay APDM 212)

8 RCT: VitD3 in patients with T1D 3 Patients with new-onset T1D (<12 weeks) Intervention 12 month: oral therapy of cholecalciferol (7IU/kg bodyweight/day) or placebo Immunologic Assessment at month, 3, 6 and 12: Immune phenotyping: FACS-analysis CD4 pos CD25 hi Foxp3 pos CD127 dim Treg Th-Subtypes (Th1, Th2, Th17) DC, B-cells, NK, NKT-cells functional tests: FACS sorted Treg and Teff ex vivo suppression co-cultures( 3 H -thymidine incorporation) Apoptosis (AnnexinV/7-AAD)

9 RCT: VitD3 in T1D % CD4 + CD25 hi CD127 - Foxp Regulatory Tcells months Placebo % suppression of Teff population < months Placebo 1 n.s %suppression 5 Placebo % suppression of Teff population months months Treiber G et al in preperation

10 RCT: VitD3 in T1D % apoptotic cells within Treg Apoptosis Treg months Placebo % mdc in Lin neg HLADR hi expressing lymphocytes Myleoid dentritic cells months Placebo No significant effect on the frequency of other peripheral immune cells Th1 /Th2 / Th17 cells B cells Cells fromtheinnateimmune system % pdc in Lin neg HLADR hi expressing lymphocytes Plasmacytoid dendritic cells months Placebo

11 RCT: VitD3 in T1D fasting C-peptide stimulated C-Peptide ng/dl p=.78 % AUC 12min 2 Placebo months p=n.s. Placebo months Insulindose Serum calcium IU/kg BW/day p= Placebo 2.6 mmol/l Placebo months months

12 Tregorigin: Thymus and Gut associated lymphoid tissue GALT Li Let al ClinImmun211

13 GALT: interfaceformaintenace ofimmune homostasis T1D: Increasedintestinal permeability precedes clinical onset of type 1 diabetes. (Bosi Diabetologia 26) Cow milk, early cereal exposure and enterovirusinfection riskfactors for islet autoimmunity. (Lempainen DMRR 212) T1D:reduced regulatory Foxp3 + T cells in duodenal biopsies (Badami Diabetes 211) IBD: Decreasedfrequency of CD4 + Foxp3 + Tregin PBMC and increased Foxp3 + cells in inflamedmucosa (Wang JDD 211). Increased apoptosis of regulatory T lymphocytes in peripheral blood and in mucosa. (VeltkampC Gut 211) VitD3 effect on Tregin gastrointestinal mucosa in humans unknown

14 VitD3 effecton Treg in gastrointestinal mucosain humans Screening 16 healthy volunteers Safety Follow-up Gastro Colo Gastro Colo 4 weeks 4weeks BL V2/V3 V4 V5/V6 V7 blood biopsy 7IU/kg BW/d Loading dose blood biopsy blood

15 Biopsy regions systematic assessment of upperandlowergi tract healthy humans 16 Age (yrs) 25±4 Females(%) 44 BMI (kg/m²) 23±3 Gating strategy FACS analyses

16 ** ** * ** ** ** ** * CD4 pos (% of CD3 pos T cells) rcentage CD8 pos (% of CD3 pos T cells) percentage Treg (% of CD4 pos T cells) percentage GC GA DD TI AO AC DC GC GA DD TI AO AC DC regions GC GA DD TI AO AC DC GC GA DD TI AO AC DC regions GC GA DD TI AO AC DC GC GA DD TI AO AC DC regions Plos one 213 percentage per percentage percentage

17 VitD3: on bloodtreg Serum 25 (OH) D 15 * ** 1 ng/ml peripheral blood regulatory T cells * * BL 8 weeks 1 weeks 8 weeks 1 weeks 8 weeks 1 weeks BL BL %CD4 + CD25 hi Foxp3 + CD127 - BL 4 weeks 8 weeks follow-up Serum 25 (OH) D 15 1 ng/ml 5 BL 4 weeks 8 weeks follow-up Helios + Treg BL 8 weeks 1 weeks %Helios + in Treg

18 1 8 6 VitD3 on GI CD4 + andcd8 + CD4 + T cells CD8 + T cells * * * BL 8 weeks %CD4 + in lymphocytes %CD8 + in lympphocytes Duodenum Illium Appendix re Colon li Colon Corpus Antrum Duodenum Illium Appendix re Colon li Colon Antrum 4 2 Corpus

19 VitD3 on GI Treg regulatory T cells BL 8 weeks Duodenum Illium Appendix re Colon li Colon Antrum Duodenum Illium Appendix re Colon li Colon Antrum Corpus Corpus %Treg in CD Baseline Helios + Treg * BL 8 weeks %Treg in CD4 %Helios + in Treg Duodenum Illium Appendix re Colon li Colon Antrum Duodenum Illium Appendix re Colon li Colon Antrum Corpus weeks VitD3 8 weeksvitd3 Corpus %Treg in CD4

20 VitD3 andmikrobiom before 8 wks VitD alters uppergi community structure Reduction of Proteobacteria Increase of richness in uppergi

21 Summary andconclusion Vitamin D3 - increases peripheral regulatory T cells in healthy humans Impaired suppressive capacity in type 1 diabetes improved with supplementation along with preservation of fasting C-peptide 8 weeksofvitd3 didnot alter total numberoftregin gastrointestinal mucosain healthyhumansbut showedan distinctpatternin Helios + Tregcompartment Vitamin D3 elevatescd8 pos cytotoxict cellnumbers, accompanied by modulation of the gut microbiome with marked reduction of Gammaproteobacteria Potential as adjunctive immunomodulatorytherapy of immunomediateddiseases like T1D in combination with other future immune therapies

22 Acknowledgements Barbara Prietl Evelyne Höller Verena Pfeifer Christine Neuper Silvia Leitgeb Anja Ribitsch CD4 + T cell Martin Tauschmann Minar Bashir Christoph Högenauer Patrizia Kump Gregor Gorkiewicz Stefan Pilz All our study participants Team Diabetes Clinics ADPP Network Research support EASD and EFSD Thank you!