York criteria, 6 RA patients and 10 age- and gender-matched healthy controls (HCs).
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1 MATERIALS AND METHODS Study population Blood samples were obtained from 15 patients with AS fulfilling the modified New York criteria, 6 RA patients and 10 age- and gender-matched healthy controls (HCs). Demographics of patients and HCs used in Figure 1B-F are shown in online supplementary table 1. Ethical permission (Oxfordshire Research Ethics Committee REC06/Q1606/139) and informed consent were obtained. T cell expansion and Th17 cell sorting PBMCs were isolated by density gradient centrifugation using Histopaque (Sigma, UK). To obtain highly purified IL-17A producing CD4+ T cells, we used an IL-17A secretion and capture assay (Miltenyi Biotec). Th17 cells from PBMCs were enriched in vitro using IL-2 (50 IU/mL, Peprotec), together with anti-cd2/3/28-coated beads (Miltenyi Biotec) for 6 days. Cells were then re-stimulated and labeled with anti-il- 17A-capture antibody followed by anti-il-17a detection and anti-cd4 (Bioloegend) according to the manufacturer s instructions (Miltenyi Biotec). CD4+IL-17A+ and CD4+IL-17A cells were sorted with a flow cytometer (FACS Aria; BD). The purity of CD4+IL-17A+ sorted cells measured by flow cytometry was > 95%. mirna sequencing and transcriptome analysis by RNA sequencing Total mirna was extracted from approximately 100,000 CD4+IL-17A+ and CD4+IL-17A- cells from 4 HCs and 4 AS patients using the mirneasy kit (Qiagen, UK) for mirna sequencing. RNA underwent quality control testing using a bionalayser followed by cdna library preparation. mir libraries were sequenced on HiSeq 2500 in a paired-end mode averaging 30 million sequenced fragments per
2 sample. Fragments were mapped to the mirbase v21 database ( ls.org/content/42/d1/d68) and differentially expressed mirs were discovered using DESeq package (Anders S and Huber W (2010). Differential expression analysis for sequence count data. Genome Biology, 11, pp. R106). Only samples with more than 12 million reads mapped to the database were considered for the differential expression analysis, which excluded one of the Th17 AS samples. mir libraries were sequenced on HiSeq 4000 averaging 36 million sequenced fragments per sample. Fragments were mapped to the human genome reference sequence GRCh37 with tophat2 (D. Kim, G. Pertea, C. Trapnell, H. Pimentel, R. Kelley, S. L. Salzberg, TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions, Genome Biol. 14, R36 (2013) ). Samples with more than 12 million reads uniquely mapped to the reference (after removing PCR duplicates) were considered for the follow-up analysis, and all eight samples passed the threshold. Gene counts were retrieved using htseq-count (HTSeq - a Python framework to work with high-throughput sequencing data, Bioinforma. Oxf. Engl. 31, (2015)) and the Gencode v19 gene annotation. Differential gene expression was analyzed with DESeq2 (M. I. Love, W. Huber, S. Anders, Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2, Genome Biol. 15, 550 (2014). Real-time polymerase chain reaction (PCR) MiScript primer assay (Qiagen) was used for semiquantitative determination of the expression of human mir-10b (MIMAT ). The primer for human mitogenactivated protein kinase kinase kinase 7 (MAP3K7) were as follows: 5 - ACTTGATGCGGTACTTTC-3 (forward) and 5 -GGTTGCGGCGATCCTA-3 (reverse). Expression of RNU6 or β-actin was used as an endogenous control. A threshold cycle (CT) was observed in the exponential phase of amplification, and
3 quantification of relative expression levels was performed using standard curves for target genes and the endogenous control. Geometric means were used to calculate the ΔΔCT (delta-delta CT) values and are expressed as 2 - ΔΔCT. The value of each control sample was set at 1, and was used to calculate the fold-change of the target genes. Purification and transfection of primary CD4+ T cells Primary CD4+ T cells were negatively isolated from PBMCs (Miltenyi Biotec). The purity was shown to be 92-97% by flow cytometry. Transfection was performed with a Neon transfection system device (Thermos Fisher Scientific, Germany) according to manufacturer s instructions. Briefly, CD4+ T cells were stimulated with 2 μg/ml Phytohaemagglutinin (PHA) before transfection with 20 μm oligonucleotides of mir- 10b mimic, mir-scramble (Qiagen), scramble-small interfering RNA (sirna), or MAP3K7-siRNA (OriGene, Germany). After transfection, cells were cultured in RPMI with 10% fetal calf serum in the presence of IL-2 (50 IU/ml), IL-7 (10 ng/ml), and anti-cd2/3/28 beads (1:20) (Miltenyi Biotec). Transfected cells were then incubated under normal conditions (humidified 37 o C/5% CO2) until analysis. The transfection efficiency was evaluated by monitoring FAM (Fluorescein) positive cells using flow cytometry, and qpcr at 24 h after transfection (Figure S1). Th17/Th1/Th2 cells differentiation and transfection of naïve CD4+ T cells Naïve CD4+ T cells were negatively isolated from PBMCs (Miltenyi Biotec). The purity was measured using flow cytometry (95-98%). Differentiation of T helper cells were carried out using 50 IU/ml IL-2, 1:5 anti-cd2/3/28 beads (Miltenyi Biotec) and lineage-skewing cytokines: 20 ng/ml IL-12 for Th1, 80 ng/ml IL-4 for Th2, 80ng/ml
4 IL-1B/IL-6/IL-23 for Th17 cells. Naïve CD4+ T cells were transfected using same approach as total CD4 transfection described above. Flow cytometry T cells were stained with CD3-BV605 and CD4-APC (BioLegend). Dead cells were excluded using Fixable Viability Dye efluor 780 (Ebioscience). Intracellular cytokine staining (ICS) of Th17 cells was carried out using BD Cytofix/Cytoperm kit (BD Bioscience). Cells were stimulated with 100ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma) and 1 μg/ml Ionomycin (Sigma) for 4 hours in the presence of GolgiSTOP and Golgi PLUG (BD Bioscience UK).) After surface staining, cells were fixed and permeabilised, then stained with IL-17A-FITC (Ebioscience). Samples were acquired on a BD LSR Fortessa instrument and FlowJo software (Tree Star) was used for analysis. Detection of cytokines in cell culture supernatants using enzyme-linked immunosorbent assays (ELISA) Transfected total CD4 or naïve CD4+ T cells were cultured for 3 days with IL-2, IL-7 and anti-cd2/3/28-coated beads (Miltenyi Biotec). Additional lineage-specific skewing cytokines were added for Th1 (20 ng/ml IL-12), Th2 (80 ng/ml IL-4) and Th17 (80ng/ml IL-1B/IL-6/IL-23) cells. Levels of IL-17A, IFN-γ and IL-4 in cell culture supernatants were assessed using ELISA according to the manufacturer s instructions (Ebioscience). Notably, for transfection of differentiating Th2 cells, IL-4 was withdrawn from cell culture after first 24 hours, allowing the use of IL-4 ELISA to measure function of Th2 cells.
5 Luciferase Activity Assay The dual-luciferase vector psicheck-map3k7-3 untranslated regions (UTR)-wild type (WT) were constructed by synthesizing the candidate seed sequences in the 3 UTRs of MAP3K7 followed by insertion of the annealed products into the psicheck-2 vector. For the mutant constructs psicheck-map3k7-3 UTR-MUT, 3-bp mutations were introduced into the seed sequences as follows: MAP3K7_3UTR WT: 5'- TCGAGTCGAC TATACCAAGTTAAAGACAGGGTA GC-3' MAP3K7_3UTR MUT: 5'- TCGAGTCGAC TATACCAAGTTAAAGACAGCCAA GC-3' For luciferase reporter assays, HEK-293T cells were seeded in 24-well plates ( /well) and transfected with 0.8 μg of the recombinant vectors in combination with 30 nm mir-10b mimics or inhibitors. Scramble-miR was used as control group. Firefly luciferase (FLuc) and Renilla luciferase (RLuc) activities were measured in cell lysates 24 h later using the dual-luciferase reporter assay system (Promega, Madison, USA). Statistical analysis Statistical analysis was performed using Prism 5.0 Software (GraphPad Software, San Diego, USA). The statistical significance of differences between means was assessed using one way Analysis of variance (ANOVA) (Figure 1B-F and Figure 2B), paired t test (Figures 2A and C, Figures 3E and F) or unpaired t test (Figures 3B and D). Mann-Whitney test (Supplementary Table) and Spearman s rank correlation coefficient were performed to test statistical dependency between two variables (Figure 3C). A p < 0.05 was considered statistically significant.
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