signature=6533feb2e7cb193e268b6b7b37509ef5,Pharmacologic Inhibition of RORγt Regulates Th17 Signatur...

Materials and Methods

Fluorescence resonance energy transfer assay

Biotinylated human RORγt protein was made at GlaxoSmithKline (GSK); biotinylated steroid receptor coactivator 1 (SRC1) was purchased from CPC Scientific; streptavidin-labeled allophycocyanin and europium were purchased from Perkin Elmer. Compounds were added into a fluorescence resonance energy transfer (FRET) mixture containing SRC1-europium and human RORγ-allophycocyanin for 1 h. The emissions at 516 and 665 nM were then read on a ViewLux in Lance mode for Europium/allophycocyanin. The percent of activation at each dose of compound was calculated and plotted using GraphPad Prism to determine the IC50s.

IL-17F promoter assay

A Jurkat cell line stably expressing RORγt and an IL-17F promoter-luciferase reporter gene were generated. The promoter sequence for the reporter is the 1075 bp of 5′ untranslated region adjacent to the start codon of the IL-17F protein coding sequence. The Jurkat cells were cultured in RPMI 1640 media (Invitrogen) containing 10% FBS (Hyclone). The Jurkat cells were diluted at 5 × 105/ml and stimulated with anti-CD3 mAb (10 μg/ml; Biolegend) in the presence of compound for 20 h. The cultures were then mixed with Steadylite luciferase detection mix (Steadylite plus Reporter Gene Assay System, Perkin Elmer) for 30 min, after which luciferase activity was read on an EnVision Multilabel Plate Reader (PerkinElmer). The percent inhibition at each concentration of compound was calculated and plotted using GraphPad Prism to determine the IC50s. We developed both IL-17A and IL-17F promoter cell lines but chose the IL-17F cell line because it gave us a better assay window for defining the structure–activity relationships of our compounds. In addition, the use of an IL-17F promoter for the first cell-based screening assay opened up the possibility of discovering RORγt inverse agonists that affect binding to both IL-17A and IL-17F promoters.

Nuclear receptor assays

The nuclear receptor ROR contains a ligand binding domain (LBD) and a DNA binding domain (DBD). Binding of an agonist or inverse agonist to the ROR-LBD induces a conformational change in the ROR-DBD that affects activity from the promoter. HEK293 cells were transiently transfected with Vector pBIND-GAL4-DBD/RORγt or RORβ or RORα-LBD and pG5 Luc, a reporter promoter containing 5 GAL4 binding sites upstream of a minimal TATA box with a luciferase reporter gene. Twenty-four hours posttransfection, compounds were added into the culture for an additional 18 h before luciferase activity was determined using an EnVision Multilabel Plate Reader (PerkinElmer). The relative light units at each concentration of compounds were plotted for IC50 determination.

T cell culture media

T cell culture medium was composed of IMDM, 2 mM l-glutamine, 0.1 mM NEAA, 1 mM Na pyruvate 1, 100 U/ml penicillin, 100 μg/ml streptomycin (all from Invitrogen) plus 10% FBS (Hyclone), and 0.05 mM 2-ME (Sigma). For naive T cell culture medium, we used X-VIVO 15 medium (LONZA) plus 2 mM l-glutamine, 1 mM Na pyruvate 1, 100 U/ml penicillin, 100 μg/ml streptomycin (all from Invitrogen). The ratio of anti-CD3/anti-CD28 Dynabead (Invitrogen) to cells was typically 1:1.

Blood, patient samples, and PBMCs

Peripheral blood buffy coats from healthy adult volunteer donors were commercially obtained from Research Blood Components. Matched peripheral blood and skin sections that underwent biopsy from patients with psoriasis undergoing surgery were commercially obtained from Tissue Solutions. Some psoriatic patient blood samples were also obtained from Bioreclamation. Human cord blood mononuclear cells were obtained as frozen stocks from All Cells. All patient samples were obtained in accordance with commercial vendor’s approved institutional review board protocols. PBMCs were prepared from buffy coats by Ficoll-Paque density grade centrifugation (GE Amersham) and extensive washing with PBS.

Lentiviral transduction

Human RORγt lentiviral stocks were produced in HEK293T cells with 10 μg DNA transfected by calcium phosphate-mediated transfection following the manufacturer’s protocol (Profection kit; Promega). Naive CD4+ or CD8+ T cells isolated from PBMCs using naive CD4+ or CD8+ T cell isolation kits (Miltenyi) were resuspended in naive T cell culture media (see the earlier T cell culture media section) at a concentration of 1 × 106 cells/ml. Anti-CD3/anti-CD28 Dynabead (Invitrogen) and RORγt-HSA or empty vector-HSA virus supernatant were added into the cells. RORγt inverse agonists were added at the time of transduction for some experiments. The culture plates were centrifuged for 30 min at 2000 rpm and then cultured under the indicated conditions for the specified number of days.

Intracellular staining

Cells used for intracellular cytokine staining were stimulated for 5 h at 37°C with PMA (10 nM) and ionomycin (1 μM) in the presence of brefeldin A (5 μg/ml) for the last 3 h (all from Sigma). Cells were fixed and permeabilized before staining for IL-17A and IL-17F. The data were acquired on a LSR FortessaII (BD Biosciences) and were analyzed using FlowJo software (Tree Star).

Naive T cell differentiation and treatment with RORγt inverse agonists

Naive CD4+ T cells were isolated using a Naive CD4 T-cell enrichment kit (Miltenyi Biotec) following manufacturer’s protocol. To study the effect of RORγt inverse agonists on Th17 differentiation, naive CD4+ T cells (1 × 106 cells/ml) were stimulated with anti-CD3/anti-CD28 Dynabeads in naive T cell culture media under Th17 polarizing conditions (20 ng/ml IL-6, 3 ng/ml TGF-β, 20 ng/ml IL-23, 20 ng/ml IL-1β, 1 μg/ml anti–IL-4 mAb, 1 μg/ml anti–IFN-γ) in the presence of compound or DMSO control. After 6 d of culture, the supernatants were harvested and cytokine concentrations were determined using Meso Scale Discovery (MSD) assays. For Th1 or Th2 cell differentiation, cells were stimulated with anti-CD3/anti-CD28 Dynabeads and cultured with IL-12 (10 ng/ml)/anti–IL-4 mAb (2 μg/ml) or IL-4 (20 ng/ml)/anti–IL-12 mAb (2 μg/ml), respectively. After 1 wk of culture, the cells were restimulated with anti-CD3/anti-CD28 Dynabeads for 24 h. The supernatants were harvested and cytokine concentrations were measured by MSD assays.

PBMCs and memory CD4+ T cells cytokine expression

PBMCs were prepared from buffy coat by Ficoll-Paque density grade centrifugation. Memory CD4+ T cells were purified using a human Memory CD4+ T cell Isolation Kit (Miltenyi Biotec). PBMCs, cord blood mononuclear cells, and memory CD4+ T cells were cultured in T cell media plus the indicated concentration of compounds. PBMCs and cord blood mononuclear cells were stimulated with soluble anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) Abs with or without IL-23 (50 ng/ml) for 5 d, and the supernatants were then harvested to detect cytokine level by MSD. Memory CD4+ T cells were stimulated with anti-CD3/anti-CD28 Dynabeads with or without IL-23 (50 ng/ml) for 2 d before the supernatants were harvested to detect cytokine level by MDS. The IC50 were calculated using XLfit software (IDBS) or GraphPad Prism.

RNA extraction, quantitative RT-PCR

Total RNA was extracted using RNeasy kits including the optional DNaseI digestion (Qiagen). cDNA synthesis and TaqMan Real Time PCR were performed as described previously (25–27). TaqMan quantitative PCR was performed on a 7900HT Real Time PCR System (Applied Biosystems). All TaqMan reagents were purchased from Applied Biosystems.

Microarray

After extraction of total RNA using an RNeasy mini kit, microarray assays were performed at the Boston University Microarray Resource Facility (Boston, MA). In brief, the RNA samples were amplified and labeled following Ambion WT Expression Kit Protocol (Life Technologies) and GeneChip Whole Transcript (WT) Sense Target Labeling Assay Manual (Affymetrix). The cRNA samples were then hybridized to Affymetrix human 1.0ST gene chips. Affymetrix data were extracted, normalized, and analyzed using GenePattern software (Broad Institute).

Imiquimod-induced cutaneous inflammation

Female BALB/c mice weighing 20 ± 2 g (10–12 wk) were purchased from Taconic Farms and housed in a pathogen-free barrier facility at Tempero Pharmaceuticals. All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed the Institutional Animal Care and Use Committee either at GSK or by the ethical review process at the institution where the work was performed. TMP778 was dissolved in the following vehicle: 3% dimethylacetamide, 10% Solutol, and 87% saline. Imiquimod (IMQ) was formulated at 50 mg/ml final concentration in ethanol/PBS/lactic acid (54:36:10%). Mice were anesthetized before applying IMQ solution on to the skin. TMP778 (20 mg/kg) or its vehicle was administered s.c. Treatment started at day 0 and continued twice a day for 10 d in the morning and afternoon with 8 h between the two doses. Ear thickness was measured daily using an engineer’s caliper (Mitutoyo) before the application of IMQ.

Histology

Mouse ears were fixed in 10% formalin, sectioned, and stained with H&E.

γδ T cell isolation

Human γδ T cells were purified from PBMCs by negative selection using EasySep Human γ/Δ T Cell Isolation Kit (Stemcell Technologies) according to the manufacturer’s protocol. The purified γδ T cells were stimulated with anti-CD3/anti-CD28 Dynabeads (1:2 cell-to-beads ratio) plus IL-1β (10 ng/ml), IL-6 (20 ng/ml), and IL-23 (50 ng/ml) for 2 wk to differentiate the cells to IL-17A–producing γδ T cells. Cells were then restimulated with anti-CD3/anti-CD28 beads plus IL-1β (10 ng/ml), IL-6 (20 ng/ml), and IL-23 (50 ng/ml) in the presence compounds for 5 d. The cytokine titers were then determined by MSD. Mouse γδ T cells were purified from draining lymph nodes and spleen of IMQ-treated mice using positive selection. Cells were stained with PE-labeled TCRγδ Ab and subsequently purified using an EasySep mouse PE selection kit (Stemcell Technologies). Cells were then treated with compound, IL-1β, and IL-23 for 20 h for gene expression analysis or 5 d for cytokine secretion by MSD.

Mononuclear cell isolation from skin sections of psoriatic patients

Five-millimeter punch biopsies from the skin of active psoriatic lesions were purchased from Tissue Solutions, U.K. The tissues were washed with buffer containing EDTA and digested with Liberase (Roche Diagnostics for 25 min at 37°C. The enzymatically digested tissues were then passed through a 70-μm filter (BD Falcon, San Jose, CA) to remove the debris. The filtered mononuclear cells were then washed twice and suspended in T cell medium. The cells were then stimulated with anti-CD3/anti-CD28 Dynabeads in a round-bottom plate and treated with compounds or DMSO as control. The supernatant was collected on day 5 to determine the IL-17A titers by MSD.

Statistics

For skin thickness, different groups were evaluated for statistical significance using the two-tailed unpaired Student t test.