11 sterol metabolism controls th17 differentiation by ......cyp51: 14-alpha sterol demethylase....

NATURE CHEMICAL BIOLOGY

Nat. Chem. Biol. 11, 141–147 (2015)

Sterol metabolism controls TH17 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 GlickIn 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 2015.

CORRECTION NOTICE

Supplementary Information

Sterol metabolism controls Th17 differentiation by generating endogenous RORγ

agonists

Xiao Hu1∗, Yahong Wang1, Ling-Yang Hao1, Xikui Liu1, Chuck A. Lesch1, Brian M.

Sanchez1, Jay M. Wendling2, Rodney W. Morgan1, Tom D. Aicher1, Laura L. Carter1, Peter

L. Toogood1 and Gary D. Glick1,3

1Lycera Corp, 2800 Plymouth Road, Building 26, Ann Arbor, MI 48109, USA.

2Seventh Wave Laboratories, 743 Spirit 40 Park Drive, Chesterfield, MO 63005, USA.

3Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.

*Correspondence to: [email protected].

Nature Chemical Biology: doi:10.1038/nchembio.1714

mailto:[email protected]

Supplementary Results


1a

Synt

hesi

sU

ptak

eEf

flux

Met

abol

ism

+10-10

Supplementary Figure 1

Th17 Treg Th1ACAT2 2.8 2.8 1.41421356

HMGCS1 6.5 8.36 4.57HMGCR 3.8 4.67 4.19

MVK 2.5 3.19 2.29PMVK 3.1 5.29 4.53

MVD 5.0 4.9 6.5IDI1 8.4 7.68 9.6

GGPS 1.0 1.0 0.5FDPS 11.8 8.62 15.45

FDFT1 5.5 5.53 7.42SQLE 8.8 8.8 1.14869835

LSS 5.3 2.7 2.29739671CYP51 7.4 7.4 7.21

TM7SF2 2.0 3.0 3.0SC4MOL 3.6 3.6 1.74110113

NSDHL 3.9 2.68 4.11HSD17B7 2.9 1.9 0.87055056

EBP 1.2 1.59 1.32SC5D 2.4 2.63901582 1.23114441

DHCR7 4.2 1.9 1.41DHCR24 7.3 5.42 4.21

LDLR 3.66 4.8 2.3VLDLR 176.8 99.34 29.98

Stab1 26.4 3.34 5.66SCARB1 2.0 1.9 1.2

ABCA1 0.04 0.09 0.31ABCG1 0.1 0.58 0.13

APOE 0.03 0.11 0.02

CYP7A1 0.17 0.48 0.91CYP7B1 0.2 0.13 0.23CYP8B1 Low Low Low

CYP11A1 77.2 27.58 19.45CYP27A1 0.18 0.22 0.57CYP39A1 0.15 0.24 0.16CYP46A1 Low Low Low

CH25H 0.14 1.62 1.00

Symbol DescriptionACAT2 Acetyl-Coenzyme A acyltransferase

HMGCS1 Hydroxymethylglutaryl-Coenzyme A synthaseHMGCR Hydroxymethylglutaryl-Coenzyme A reductase

MVK Mevalonate kinasePMVK Phosphomevalonate kinase

MVD Mevalonate decarboxylase

IDI1 Isopentenyl-diphosphate delta isomerase

GGPS Geranylgeranyl diphosphate synthase FDPS Farnesyl diphosphate synthetase

FDFT1 Farnesyl diphosphate farnesyl transferaseSQLE Squalene epoxidase

LSS Lanosterol synthaseCYP51 14-alpha sterol demethylase

TM7SF2 3Beta-hydroxysterol Delta(14)-reductase SC4MOL Methylsterol monooxygenase

NSDHL NAD(P) dependent steroid dehydrogenase-likeHSD17B7 Hydroxysteroid (17-beta) dehydrogenase 7

EBP Emopamil binding protein (sterol isomerase)SC5D Sterol-C5-desaturase

DHCR7 7-dehydrocholesterol reductaseDHCR24 24-dehydrocholesterol reductase



1b

2b

2a

Veh Ketoconazole

43% 20%

Keto + Lano Keto + Zymo

18% 30%

IL-17A

CD4

*

*

*


* **

2c

2d

*

2e

2f


3a 3b

3c


% B

as

al

Ac

tiv

ity


4a

4b

*

Supplementary Figure 4R

elat

ive

Expr

essi

on

4d

4c

IL-1

7AIL

-17A

Treg

Th1

FOXP3

IFNγ

15% 15% 11%

15% 16% 14%

IL-1

7ATh17

RORγt

50% 44% 49%

10% 1% 5%

Veh Urso Urso + Desmo

4e



SterolsEC50(µM)

Cholesterol sulfate 525-OHC sulfate 0.5Desmosterol sulfate 0.55α,6α-Epoxycholestanol sulfate 0.1Pregnenolone sulfate >10DHEA sulfate >10Cholesterol palmitate >10Cholesterol acetate >10DHEA >10Pregnenolone >10Calcitriol >10Cholecaciferol (Vitamin D3) >107α, 25-diOHC >107α, 27-diOHC 520R,22R-diOHC >105α,6α-Epoxycholestanol 324S,25-Epoxycholesterol 0.17α-OHC 120α-OHC 0.522R-OHC 0.124S-OHC 0.225-OHC 0.127-OHC 0.1

4g4f


5d

5b

5c

0.001 0.01 0.1 1 10 1000

50

100

150

200

250CholesterolCholesterol-sulfate

Concentration [µM]

% B

asal

Act

ivity

0.001 0.01 0.1 1 10 1000

50

100

150

200

250 Desmosterol-SulfateDesmosterol

Concentration [µM]

% B

asal

Act

ivity


0.001 0.01 0.1 1 10 1000

50

100

150

200

25025-OHC25-OHC-sulfate

Concentration [µM]

% B

asal

Act

ivity

5a

5e

5f

Sterol sulfate tested ng /106 cells25-Hydroxycholesterol sulfate ND (<0.0003)

5α,6α-Epoxycholestanol sulfate ND (<0.0003)Desmosterol sulfate 0.003 ± 0.001Cholesterol sulfate 2.875 ± 0.033

5g


***


25X

5h

5k

5i

Gal4-LXRβ

0.1 1 10 1000

100

200

300

400

500100020003000 Desmo-Sulfate

Chole-SulfateGW3965

Concentration [µM]

% B

asal

Act

ivity

5j

*


6c

RORg VCKSYRETCQLRLEDLLR...FAKRLSGFMELCQNDQIVLLKAGAMEVVLVRMCRAYNADNRTV 376

RORa ISKSHLETCQYLREELQQ...FAKRIDGFMELCQNDQIVLLKAGSLEVVFIRMCRAFDSQNNTV 379

RORb IIKSHLETCQYTMEELHQ...FAKRITGFMELCQNDQILLLKSGCLEVVLVRMCRAFNPLNNTV 320

LXRa LVAAQQQCNRRSFSDRLR...FAKQLPGFLQLSREDQIALLKTSAIEVMLLETSRRYNPGSESI 313

LXRb LVAAQLQCNKRSFSDQPK...FAKQVPGFLQLGREDQIALLKASTIEIMLLETARRYNHETECI 327

6d

LXRβ - 24-EpoxycholesterolRORα - cholesterol sulfate

*

*


6a 6b High TCR activation

Low TCR activation


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 CYP3A41, 2. However,

a non-azole CYP3A4 inhibitor mifepristone3 or a well-known PXR activator rifampicin1

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


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)


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 < 0.008 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 < 0.005 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-


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.


References cited in supplementary materials

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(arylcarbonyl)thiazol-2-yl)amides and N-(5-(arylcarbonyl)thiophen-2-yl)amides as potent RORgammat inhibitors. Bioorganic & medicinal chemistry 2014, 22(2): 692-702.

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Ligand binding domain in complex with cholesterol sulfate at 2.2 A. The Journal of biological chemistry 2004, 279(14): 14033-14038.

6. Williams S, Bledsoe RK, Collins JL, Boggs S, Lambert MH, Miller AB, et al. X-ray crystal structure

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11 sterol metabolism controls th17 differentiation by ......cyp51: 14-alpha sterol demethylase....

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