CORRECTION NOTICE. Nat. Chem. Biol. 11, (2015) Sterol metabolism controls T H 17 differentiation by generating endogenous RORγ agonists

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1 CORRECTION NOTICE Nat. Chem. Biol. 11, (2015) Sterol metabolism controls T H 17 differentiation by generating endogenous RORγ agonists Xiao Hu, Yahong Wang, Ling-Yang Hao, Xikui Liu, Chuck A Lesch, Brian M Sanchez, Jay M Wendling, Rodney W Morgan, Tom D Aicher, Laura L Carter, Peter L Toogood & Gary D Glick In the version of this supplementary file originally posted online, the zymosterol and zymostenol structures shown in Supplementary Figure 1b were depicted with a double bond at C14-C15, where there should have been a single bond. The error has been corrected in this file as of 15 July NATURE CHEMICAL BIOLOGY

2 Supplementary Information Sterol metabolism controls Th17 differentiation by generating endogenous RORγ agonists Xiao Hu 1, Yahong Wang 1, Ling-Yang Hao 1, Xikui Liu 1, Chuck A. Lesch 1, Brian M. Sanchez 1, Jay M. Wendling 2, Rodney W. Morgan 1, Tom D. Aicher 1, Laura L. Carter 1, Peter L. Toogood 1 and Gary D. Glick 1,3 1 Lycera Corp, 2800 Plymouth Road, Building 26, Ann Arbor, MI 48109, USA. 2 Seventh Wave Laboratories, 743 Spirit 40 Park Drive, Chesterfield, MO 63005, USA. 3 Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA. Correspondence to: hu@lycera.com.

3 Supplementary Results

4 Supplementary Figure 1 1a Synthesis Uptake Efflux Metabolism Th17 Treg Th1 ACAT HMGCS HMGCR MVK PMVK MVD IDI GGPS FDPS FDFT SQLE LSS CYP TM7SF SC4MOL NSDHL HSD17B EBP SC5D DHCR DHCR LDLR VLDLR Stab SCARB CYP7A CYP7B CYP8B1 Low Low Low CYP11A CYP27A CYP39A CYP46A1 Low Low Low CH25H ABCA ABCG APOE Symbol Description ACAT2 Acetyl-Coenzyme A acyltransferase HMGCS1 Hydroxymethylglutaryl-Coenzyme A synthase HMGCR Hydroxymethylglutaryl-Coenzyme A reductase MVK Mevalonate kinase PMVK Phosphomevalonate kinase MVD Mevalonate decarboxylase IDI1 Isopentenyl-diphosphate delta isomerase GGPS Geranylgeranyl diphosphate synthase FDPS Farnesyl diphosphate synthetase FDFT1 Farnesyl diphosphate farnesyl transferase SQLE Squalene epoxidase LSS Lanosterol synthase CYP51 14-alpha sterol demethylase TM7SF2 3Beta-hydroxysterol Delta(14)-reductase SC4MOL Methylsterol monooxygenase NSDHL NAD(P) dependent steroid dehydrogenase-like HSD17B7 Hydroxysteroid (17-beta) dehydrogenase 7 EBP Emopamil binding protein (sterol isomerase) SC5D Sterol-C5-desaturase DHCR7 7-dehydrocholesterol reductase DHCR24 24-dehydrocholesterol reductase

5 Supplementary Figure 1 1b

6 Supplementary Figure 2 2a 2d 2b Veh Ketoconazole 2e Keto + Lano 43% 20% Keto + Zymo 2f CD4 18% 30% IL-17A 2c

7 Supplementary Figure 3 3a 3b % Basal Activity 3c

8 Supplementary Figure 4 4a 4d Th17 IL-17A Veh Urso Urso + Desmo 10% 1% 5% 50% 44% 49% RORγt Treg 4b 4c Relative Expression IL-17A 15% 15% 11% FOXP3 Th1 IL-17A 15% 16% 14% IFNγ 4e

9 Supplementary Figure 4 4f 4g EC 50 Sterols (µm) Cholesterol sulfate 5 25-OHC sulfate 0.5 Desmosterol sulfate 0.5 5α,6α-Epoxycholestanol sulfate 0.1 Pregnenolone sulfate >10 DHEA sulfate >10 Cholesterol palmitate >10 Cholesterol acetate >10 DHEA >10 Pregnenolone >10 Calcitriol >10 Cholecaciferol (Vitamin D3) >10 7α, 25-diOHC >10 7α, 27-diOHC 5 20R,22R-diOHC >10 5α,6α-Epoxycholestanol 3 24S,25-Epoxycholesterol 0.1 7α-OHC 1 20α-OHC R-OHC S-OHC OHC OHC 0.1

Supplementary Figure 5 5a 5b % Basal Activity

Cholesterol-sulfate % Basal Activity 250 200

01 0.1 1 10 100 Concentration [µm] 0 0.001 0.

01 0.1 1 10 100 Concentration [µm] 5f 5g

10 Supplementary Figure 5 5a 5b % Basal Activity Cholesterol Cholesterol-sulfate % Basal Activity OHC 25-OHC-sulfate 5c Concentration [µm] Concentration [µm] 5d % Basal Activity Desmosterol-Sulfate Desmosterol 5e Concentration [µm] 5f 5g Sterol sulfate tested ng /10 6 cells 25-Hydroxycholesterol sulfate ND (<0.0003) 5α,6α-Epoxycholestanol sulfate ND (<0.0003) Desmosterol sulfate ± Cholesterol sulfate ± 0.033

3000 Desmo-Sulfate 2000 Chole-Sulfate 1000

11 Supplementary Figure 5 5h 5i 25X 5j % Basal Activity Gal4-LXRβ 3000 Desmo-Sulfate 2000 Chole-Sulfate GW Concentration [µm] 5k

Supplementary Figure 6 6a 6b High TCR activation

..FAKRLSGFMELCQNDQIVLLKAGAMEVVLVRMCRAYNADNRTV 376

..FAKRIDGFMELCQNDQIVLLKAGSLEVVFIRMCRAFDSQNNTV 379

..FAKRITGFMELCQNDQILLLKSGCLEVVLVRMCRAFNPLNNTV 320

..FAKQLPGFLQLSREDQIALLKTSAIEVMLLETSRRYNPGSESI 313

12 Supplementary Figure 6 6a 6b High TCR activation Low TCR activation 6c 6d RORg VCKSYRETCQLRLEDLLR...FAKRLSGFMELCQNDQIVLLKAGAMEVVLVRMCRAYNADNRTV 376 RORa ISKSHLETCQYLREELQQ...FAKRIDGFMELCQNDQIVLLKAGSLEVVFIRMCRAFDSQNNTV 379 RORb IIKSHLETCQYTMEELHQ...FAKRITGFMELCQNDQILLLKSGCLEVVLVRMCRAFNPLNNTV 320 LXRa LVAAQQQCNRRSFSDRLR...FAKQLPGFLQLSREDQIALLKTSAIEVMLLETSRRYNPGSESI 313 LXRb LVAAQLQCNKRSFSDQPK...FAKQVPGFLQLGREDQIALLKASTIEIMLLETARRYNHETECI 327 RORα - cholesterol sulfate LXRβ - 24-Epoxycholesterol

13 Supplementary Figure Legends Fig. 1. Cholesterol synthesis and metabolism pathways. (a) Left, cholesterol synthetic and uptake pathways are induced while metabolism and efflux pathways are decreased in Treg and Th1 cells. Right, a description of genes involved in cholesterol synthesis. (b) Top, numbering system for sterols. Bottom, cholesterol synthetic pathways with structures showing precursors after cyclization. Inhibitors are indicated in red. Fig. 2. Inhibiting cholesterol synthesis reduces Th17 differentiation. (a) Statins decrease IL-17 production. Simvastatin is at 10 and 2 µm. Atorvastatin is at 2 µm. (b) Ketoconazole (10 µm) reduces Th17 differentiation. Zymosterol but not lanosterol rescues Th17 differentiation. (c) Ketoconazole decreases IL-17A, IL-17F and IL-23R but not RORγt (RORC2), IFNγ and IL-21 mrna expression. (d) Ketoconazole decreases IL-17A production when T cells are activated by a specific antigen. OTII splenocytes are differentiated into Th17 in the presence of 500 ng/ml OVA peptide and Th17 polarizing cytokines., p < 0.05 vs. vehicle (Veh). (e) CYP3A4 inhibitor mifepristone or PXR activator rifampicin does not inhibit IL-17A production. Compounds were at 10 µm. Ketoconazole, econazole and clotrimazole activate PXR and inhibit CYP3A4 1, 2. However, a non-azole CYP3A4 inhibitor mifepristone 3 or a well-known PXR activator rifampicin 1 did not significantly decrease IL17 production, confirming that these azole based inhibitors decrease Th17 differentiation through inhibiting CYP51. (f) Desmosterol does not affect T- bet mrna expression in Th1 cells. Fig. 3. Select sterols are RORγ agonists. (a) Representative TR-FRET data showing increase of coactivator recruitment by sterols in the presence of RORγ antagonist ursolic

14 acid (left) or digoxin (right). (b) Ketoconazole (10 µm) and RORγ antagonist ursolic acid (2 µm) reduce Gal4-RORγ activity. (c) Azoles do not block coactivator recruitment by RORγ. Fig. 4. Select sterols are RORγ agonists in Th17 cells. (a) Sterols (15 µm) increase IL- 17A production in the presence of ketoconazole (10 µm)., p < 0.01 vs. Ketoconazole. (b) Desmosterol increases IL-17A production in the presence of digoxin (15 µm) or a synthetic RORγ antagonist (compound 192 at 100 nm) 4. (c) Desmosterol increases the expression of RORγ target genes (IL17F and IL23R) but not non-target genes (IL21 and IFNγ). (d) Desmosterol increase IL-17A but has no effects on RORγ in Th17 cells, FOXP3 in Treg cells or IFNγ in Th1 cells. Intracellular staining was done after 4 hours of re-stimulation with PMA/Ionomycin/Brefeldin. (e) Desmosterol increases IL-17A in Th17 cells but not in Treg or Th1 cells. (f) CYP11A1 products, 20-OHC and 22R-OHC but not pregenenolone can activate RORγ. (g) Some sterol conjugates and derivatives can increase RORγ coactivator recruitment. Assay was done in the presence of 2 µm ursolic acid. Fig. 5. Sterol sulfates activate RORγ but not LXRβ. (a) Schematic diagram of sterol sulfate biosynthesis. (b) Cholesterol sulfate and 25-OHC sulfate activate RORγ in the presence of ursolic acid (2 µm) in a coactivator recruitment assay. (c) Sterol sulfates strongly activate Gal4-RORγ in the presence of ursolic acid (2 µm) or ketoconazole (10 µm). (d) Desmosterol sulfate increases coactivator recruitment in the absence of ursolic acid. (e) Viability of cells in the presence various inhibitors for 4 days (4d) during Th17 differentiation or during last day of differentiation (1d). Sterols were at 15 µm. (f)

15 Desmosterol sulfate does not increase IFNγ mrna expression. (g) Sterol sulfate levels in Th17 cells. (h) Cholesterol efflux is lower in Th17 cells vs. naïve CD4+ T cells and LXR agonist GW3965 (1 µm) enhances efflux in Th17 cells. Cholesterol efflux was measured using TopFluor (BODIPY) cholesterol., p < vs. naïve CD4+ T cells or Th17+ GW3965. (i) ABCA1 expression decreases during Th17 differentiation. (j) Sterol sulfates do not activate LXRβ. (k) Inhibition of sulfate formation partially induced ABCG1 expression. Chlorate is an inhibitor of adenosine 3'-phosphate 5'-phosphosulfate (PAPS) synthesis. PAPS is a universal sulfate donor which provides sulfate moiety for sterol sulfate conjugation., p = 0.05, p = 0.004, vs. vehicle (Veh). Fig. 6. Sterol sulfates are also RORα agonists. (a) Ketoconazole decreases cell viability at 30 µm concentrations but not at 10 µm concentrations. Cell viability were assayed using Alamar Blue cell viability dye. Relative viability was calculated by normalizing to the control group without ketoconazole. (b) Desmosterol increases IL-17A production under high TCR or low TCR activation conditions. Splenocytes from OTII mice were differentiated in the presence of 500 (high TCR activation) or 50 ng/ml (Low TCR activation) OVA peptide alone with Th17 polarizing cytokines TGFβ, IL-6 and IL-1β. (c) Desmosterol sulfate and cholesterol sulfate (15 µm) can activate RORα in the presence of ketoconazole (10 µm) in Gal4-RORα assay., p < vs. ketoconazole. (d) Top, Sequence alignment of RORs and LXRs showing the residues surrounding sterol ligands. The RORα, RORβ, and RORγ sequences are conserved around key amino acids (in red) which bind to the cholesterol sulfate in RORα. Bottom, Crystal structures of RORγ complexed with cholesterol sulfate (1S0X) 5 and LXRβ complexed with 24S,25-

16 epoxycholesterol (1P8D) 6. In RORα, Q289, Y390 and R370 make hydrogen bonds to the sulfate moiety. In addition, positively charged R367 also makes a hydrogen bond through one water molecule with the negatively charged sulfate. In LXR, the residue corresponding to RORα R367 is replaced with a negatively charged glutamate (E315). A371 in RORα is replaced by R319 in LXR. R319 makes an intra-molecular hydrogen bond with E315 and the guanidine side-chain occupies the same location as the sulfate moiety of cholesterol sulfate in the RORα structure. In order for a sterol sulfate to bind to LXR, substantial rearrangement of the receptor must occur and/or the sterol moiety must shift several angstroms, which may explain the lack of activation by sterol sulfates on LXR.

17 References cited in supplementary materials 1. Svecova L, Vrzal R, Burysek L, Anzenbacherova E, Cerveny L, Grim J, et al. Azole antimycotics differentially affect rifampicin-induced pregnane X receptor-mediated CYP3A4 gene expression. Drug metabolism and disposition: the biological fate of chemicals 2008, 36(2): Monostory K, Hazai E, Vereczkey L. Inhibition of cytochrome P450 enzymes participating in p- nitrophenol hydroxylation by drugs known as CYP2E1 inhibitors. Chemico-biological interactions 2004, 147(3): He K, Woolf TF, Hollenberg PF. Mechanism-based inactivation of cytochrome P-450-3A4 by mifepristone (RU486). The Journal of pharmacology and experimental therapeutics 1999, 288(2): Wang Y, Cai W, Zhang G, Yang T, Liu Q, Cheng Y, et al. Discovery of novel N-(5- (arylcarbonyl)thiazol-2-yl)amides and N-(5-(arylcarbonyl)thiophen-2-yl)amides as potent RORgammat inhibitors. Bioorganic & medicinal chemistry 2014, 22(2): Kallen J, Schlaeppi JM, Bitsch F, Delhon I, Fournier B. Crystal structure of the human RORalpha Ligand binding domain in complex with cholesterol sulfate at 2.2 A. The Journal of biological chemistry 2004, 279(14): Williams S, Bledsoe RK, Collins JL, Boggs S, Lambert MH, Miller AB, et al. X-ray crystal structure of the liver X receptor beta ligand binding domain: regulation by a histidine-tryptophan switch. The Journal of biological chemistry 2003, 278(29):

Supplementary Materials Modeling cholesterol metabolism by gene expression profiling in the hippocampus

Supplementary Materials Modeling cholesterol metabolism by gene expression profiling in the hippocampus Christopher M. Valdez 1, Clyde F. Phelix 1, Mark A. Smith 3, George Perry 1,2, and Fidel Santamaria