RORγt

The RORγt reporter's activity was considerably decreased after treatment with 3-Oxo-5β-cholanoic acid. TH17 cell differentiation is likely inhibited by 3-Oxo-5β-cholanoic acid by physical interaction with RORγt and inhibition of its transcriptional activity, as suggested by these data[1].

3-Oxo-5β-cholanoic acid (Dehydrolithocholic acid) (0.3% (w/w); po; for 1 week) decreases the percentage of TH17 cells in the ileum significantly[1].

Mammalian Luciferase Reporter Assay[1]
Reporter assays were conducted as previously described14. Briefly, 50,000 human embryonic kidney 293 cells per well were plated in 96-well plates in antibiotic-free Dulbecco’s Modified Eagle Media (DMEM) containing 1% fetal calf serum (FCS). Cells were transfected with a DNA mixture containing 0.5 μg/mL of firefly luciferase reporter plasmid, 2.5 ng/mL of a plasmid containing Renilla luciferase, and Gal4-DNA binding domain-RORγ (0.2 μg/mL). Transfections were performed using TransIT-293 according to the manufacturer’s instruction. Bile acids or vehicle control were added 24h after transfection and luciferase activity was measured 16 h later using the dual-luciferase reporter kit.

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Microscale Thermophoresis Assay[1]
The binding affinity of the compounds with RORγ ligand-binding domain (LBD) was analyzed by microscale thermophoresis (MST). Purified RORγ-LBD was labeled with the Monolith NT™ Protein Labeling Kit RED. Serially-diluted compounds, with concentrations of 1 mM to 20 nM, were mixed with 55 nM labeled RORγ-LBD at room temperature and loaded into Monolith TM standard-treated capillaries. Binding was measured by monitoring the thermophoresis with 20% LED power and ‘Medium’ MST power on a Monolith NT.115 instrument with the following time setting: 5s Fluo, Before; 20s MST On; and 5s Fluo, After. Kd values were fitted using the NT Analysis software (Nano Temper Technologies).

In Vitro T Cell Culture[1]
Naïve CD4+ (CD62L+ CD44− CD25− CD4+) T cells were isolated from the spleens and the lymph nodes of mice of designated genotypes with FACS sorting. For certain experiments, naïve CD4+ T cells were enriched using naïve CD4+ T cell isolation kits. Naïve CD4+ T cells (40,000 cells) were cultured in a 96-well plate pre-coated with hamster IgG in T cell medium (RPMI, 10% fetal bovine serum, 25 mM glutamine, 55 µM 2-mercaptoethanol, 100 U/mL penicillin, 100 mg/mL streptomycin) supplemented with 0.25 µg/mL anti-CD3 (clone 145–2C11) and 1 µg/mL anti-CD28 (clone 37.51). For Th0 culture, T cells were cultured with the addition of 100 U/mL of IL-2. For Th1 cell differentiation, T cells were cultured with the addition of 100 U/mL of IL-2, 10 µg/mL of anti-IL-4 (clone 11B11) and 10 ng/mL of IL-12. For Th2 cell differentiation, T cells were cultured with the addition of 10 µg/mL of anti-IFNγ (clone XMG1.2) and 10 ng/mL of IL-4. For Th17 cell differentiation, T cells were cultured with the addition of 10 ng/mL of IL-6 and 0.5 ng/mL of TGF-β. For Treg culture, T cells were cultured with the addition of 100 U/mL of IL-2 and various concentrations of TGF-β. For most in vitro experiments to test the effects of isoalloLCA, no additional TGF-β was added. Bile acids, retinoic acid or mitoQ, or mitoPQ were added either at 0 or 16h time points. Compounds with low water solubility were sonicated before adding to the culture. Cells were harvested and assayed by flow cytometry on day 3. For ROS and mitochondrial membrane potential detection, cells cultured for 2 days were incubated with 5 µM of mitoSOX , 10 µM of DCFDA or 2 µM of JC-1 for 30 min and assayed with flow cytometry. Flow Cytometry[1]
Cells harvested from in vitro culture or in vivo mice experiments were stimulated with 50 ng/mL PMA (Phorbol 12-myristate 13-acetate) and 1 µM ionomycin in the presence of GolgiPlug for 4h to determine cytokine expression. After stimulation, cells were stained with cell surface marker antibodies and LIVE/DEAD Fixable dye, Aqua, to exclude dead cells, fixed and permeabilized with a FoxP3/Transcription factor staining kit, followed by staining with cytokine- and/or transcription factor-specific antibodies. All flow cytometry analyses were performed on an LSR II flow cytometer and data were analyzed with FlowJo software).

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Cell Proliferation Assay[1]
Naïve CD4+ T cells were labeled with 1 µM carboxyfluorescein succinimidyl ester and cultured for 3 days prior to FACS analysis.

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In Vitro Suppression Assay[1]
A total of 2.5 × 104 freshly-purified naïve CD4+CD25−CD44−CD62Lhigh T cells from CD45.1 B6 mice were labeled with 1 µM CFSE, activated with soluble anti-CD3 (1 µg/mL) and 5 × 104 APCs in 96-well round-bottom plates for 3 days in the presence of tester cells (CD45.2). The CFSE dilution of CD45.1 Tconv cells was assessed by flow cytometry.

Animal/Disease Models: B6 Jax mice (with a faecal slurry containing SFB)[1]
Doses: 0.3% (w/w)
Route of Administration: Gavaged; for 1 week
Experimental Results: Dramatically decreased the percentage of ileal TH17 cells.

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In vivo bile acid analysis[1]
Stock solutions of all bile acids were prepared by dissolving the compounds in molecular biology grade DMSO. These solutions were used to establish standard curves. Glycocholic acid or β-muricholic acid (β-MCA) was used as the internal standard for mouse and human samples, respectively. Bile acids were extracted from mouse cecal and human fecal samples and quantified by Ultra-High Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) as previously reported49. The limits of detection of individual bile acids in tissues (in picomol/mg wet mass) are as follows: βMCA, 0.10; isoalloLCA, 0.45; isoLCA, 0.29; LCA, 0.12; alloLCA, 0.43; and 3-oxoLCA, 0.18.

[1]. Bile acid metabolites control TH17 and Treg cell differentiation. Nature. 2019 Dec;576(7785):143-148.

3-oxo-5beta-cholanic acid is an oxo-5beta-cholanic acid. It is a conjugate acid of a 3-oxo-5beta-cholanate.

C24H38O3

374.55672

374.282

1553-56-6

5283906

White to off-white solid powder

1.069 g/cm3

509.3ºC

275.9ºC

0mmHg at 25°C

1.52

5.715

1

3

4

27

613

8

OC(CC[C@H]([C@H]1CC[C@H]2[C@@H]3CCC4CC(CC[C@]4(C)[C@H]3CC[C@]12C)=O)C)=O

KIQFUORWRVZTHT-OPTMKGCMSA-N

InChI=1S/C24H38O3/c1-15(4-9-22(26)27)19-7-8-20-18-6-5-16-14-17(25)10-12-23(16,2)21(18)11-13-24(19,20)3/h15-16,18-21H,4-14H2,1-3H3,(H,26,27)/t15-,16-,18+,19-,20+,21+,23+,24-/m1/s1

(4R)-4-[(1R,3aS,3bR,5aR,9aS,9bS,11aR)-9a,11a-dimethyl-7-oxo-hexadecahydro-1H-cyclopenta[a]phenanthren-1-yl]pentanoic acid

Dehydrolithocholic acid; 3-Oxocholanic acid; Dehydrolithocholic acid; 1553-56-6; 3-Ketolithocholic acid; 3-Oxo-5beta-cholanoic Acid; 3-keto-lithocholic acid; 3-Oxocholan-24-oic acid; 3-Ketolithocholic acid; 3-Oxolithocholic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 100 mg/mL (266.98 mM)
H2O: < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.67 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.67 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.67 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 3.33 mg/mL (8.89 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication (<60°C).
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)