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
Supplementary Results
Nature Chemical Biology: doi:10.1038/nchembio.1714
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
Nature Chemical Biology: doi:10.1038/nchembio.1714
Supplementary Figure 1
1b
2b
2a
Veh Ketoconazole
43% 20%
Keto + Lano Keto + Zymo
18% 30%
IL-17A
CD4
*
*
*
Supplementary Figure 2
* **
2c
2d
*
2e
2f
Nature Chemical Biology: doi:10.1038/nchembio.1714
3a 3b
3c
Supplementary Figure 3
% B
as
al
Ac
tiv
ity
Nature Chemical Biology: doi:10.1038/nchembio.1714
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
Nature Chemical Biology: doi:10.1038/nchembio.1714
Supplementary Figure 4
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
Nature Chemical Biology: doi:10.1038/nchembio.1714
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
Supplementary Figure 5
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
Nature Chemical Biology: doi:10.1038/nchembio.1714
***
Supplementary Figure 5
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
*
Nature Chemical Biology: doi:10.1038/nchembio.1714
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
*
*
Supplementary Figure 6
6a 6b High TCR activation
Low TCR activation
Nature Chemical Biology: doi:10.1038/nchembio.1714
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
Nature Chemical Biology: doi:10.1038/nchembio.1714
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)
Nature Chemical Biology: doi:10.1038/nchembio.1714
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-
Nature Chemical Biology: doi:10.1038/nchembio.1714
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.
Nature Chemical Biology: doi:10.1038/nchembio.1714
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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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Nature Chemical Biology: doi:10.1038/nchembio.1714
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