Presentation_1_OTUD6B-AS1 Might Be a Novel Regulator of Apoptosis in Systemic Sclerosis.pptx

Antisense long non-coding RNAs (AS lncRNAs) have increasingly been recognized as important regulators of gene expression and they have been found to play key roles in several diseases. However, very little is known about the role of AS lncRNAs in fibrotic diseases such as systemic sclerosis (SSc). Our recent screening experiments by RNA sequencing showed that ovarian tumor domain containing 6B antisense RNA1 (OTUD6B-AS1) and its sense gene OTUD6B were significantly downregulated in SSc skin biopsies. Therefore, we aimed to identify key regulators of OTUD6B-AS1 and to analyze the functional relevance of OTUD6B-AS1 in SSc. OTUD6B-AS1 and OTUD6B expression in SSc and healthy control (HC) dermal fibroblasts (Fb) after stimulation with transforming growth factor-β (TGFβ), Interleukin (IL)-4, IL-13, and platelet-derived growth factor (PDGF) was analyzed by qPCR. To identify the functional role of OTUD6B-AS1, dermal Fb or human pulmonary artery smooth muscle cells (HPASMC) were transfected with a locked nucleic acid antisense oligonucleotide (ASO) targeting OTUD6B-AS1. Proliferation was measured by BrdU and real-time proliferation assay. Apoptosis was measured by Caspase 3/7 assay and Western blot for cleaved caspase 3. While no difference was recorded at the basal level between HC and SSc dermal Fb, the expression of OTUD6B-AS1 and OTUD6B was significantly downregulated in both SSc and HC dermal Fb after PDGF stimulation in a time-dependent manner. Only mild and inconsistent effects were observed with TGFβ, IL-4, and IL-13. OTUD6B-AS1 knockdown in Fb and HPASMC did not affect extracellular matrix or pro-fibrotic/proinflammatory cytokine production. However, OTUD6B-AS1 knockdown significantly increased Cyclin D1 expression at the mRNA and protein level. Moreover, silencing of OTUD6B-AS1 significantly reduced proliferation and suppressed apoptosis in both dermal Fb and HPASMC. OTUD6B-AS1 knockdown did not affect OTUD6B expression at the mRNA level and protein level. Our data suggest that OTUD6B-AS1 regulates proliferation and apoptosis via cyclin D1 expression in a sense gene independent manner. This is the first report investigating the function of OTUD6B-AS1. Our data shed light on a novel apoptosis resistance mechanism in Fb and vascular smooth muscle cells that might be relevant for pathogenesis of SSc.</p

Effect of compounds on CD86 expression in primary CD19+ B cells.

<p>Purified human CD19+ primary B cells were incubated with 10 ng/ml IL4 alone (“No activation”) or IL4+64 ng/ml tCD40L (“Activation”), together with different concentrations of drugs for 48 hours. CD86 expression was measured by PE GeoMFI on CD19+ gated B cells. The chemical structure of each compound is shown.</p

Relative IC₅₀ for two “known” and two “novel” compounds in both BL2-NFκB-Luc and Ramos-NFκB-Luc cell lines.

<p>Relative IC₅₀ is the concentration required to bring the dose-response curve to the halfway point between the top and bottom plateaus of the curve.</p

CD40 knockdown and CD40-luciferase assay in BL2 cells.

<p>(A) Schematic of the canonical CD40 – NF-|B signaling pathway in B cells. (B) RNAi perturbation of <i>CD40</i> in two distinct clones derived from BL2 cells decreases CD40 protein levels by 55% (left) and 40% (middle) compared to the BL2 parent line (black, right); (C) More CD40 on the surface of BL2 cells increases RelA (p65) phosphorylation following activation with tCD40L, as measured by Western blot, with maximum activation at 15 minutes. Results are shown for the same two shRNA lines and parental BL2 cell line as in (B). This is a representative example of multiple experiments. (D) Titration of tCD40L leads to increased luciferase activity. Each experiment was performed in triplicate. The red circle represents ∼80% maximum luciferase activity (64 ng/ml tCD40L). Luciferase activity at baseline (i.e., no tCD40L activation) was subtracted from each measurement to plot results. (E) Titration of IKK inhibitor VII leads to inhibition of luciferase activity following tCD40L activation. Each experiment was performed in duplicate. (F) The luciferase assay is robust, with Z'-factor>0.80 and >60-fold inhibition of luciferase activity without killing cells across different plates.</p

Genetic data on risk of RA and CD40 protein levels.

<p>(A) The regional association plot from analysis of Immunochip (iChip) data in 7,222 CCP+ cases and 15,870 controls. Gene location is shown along the bottom of the graph, with observed –log(P) value along the left Y-axis and recombination rate along the right Y-axis. Each SNP is plotted is a circle, with color scheme (red to white) in reference to the extent of linkage disequilibrium with the index SNP, rs4810485 (labeled as a diamond). (B) The regional association plot from analysis of iChip data and CD40 protein levels in 90 healthy control individuals. (C) A box-whisker's plot of SNP (rs4810485) and CD40 protein levels in B cells from healthy control individuals, where T = non-risk allele and G = risk allele. (D) A box-whisker's plot of SNP (rs4810485) and <i>CD40</i> mRNA levels in PBMC's from two separate collections (total of 1,441 healthy control individuals); T = non-risk allele and G = risk allele.</p

Percent maximum inhibition for two “known” and two “novel” compounds in both BL2-NFκB-Luc and Ramos-NFκB-Luc cell lines.

<p>For each compound, we calculate the maximum amount of inhibition observed at the highest concentration of drug relative to zero luciferase activity.</p

Small molecule screen of CD40-mediated NF-kB signaling in BL2 cells.

<p>(A) Results from duplicate experiments screening 1,982 compounds. Red circles are our positive control (IKK inhibitor VII); grey circles are our neutral controls (DMSO only); and blue circles are test compounds. The red dashed line indicates >2SD from the mean of the neutral controls, which defines our “hit” compounds (n = 81 compounds). (B) Dose-response curves for two compounds known to inhibit inflammation [CID = 5282230 (tranilast)] or NF-|B signaling [CID = 5282360 (4-hydroxy-estradiol)] in the BL2-NF|B-Luc cell lines. (C) Dose-response curves for two compounds not previously implicated in inflammation, NF-κB signaling, CD40 signaling, or other biological pathways related to rheumatoid arthritis: CID = 306804, [4-(1-acetyl-4-oxo-2H-3,1-benzoxazin-2-yl)phenyl] acetate; and CID = 7309015, 8-[(Z)-3-(3,4-dimethoxyphenyl)prop-2-enoyl]-7-hydroxy-4-methylchromen-2-one. Red line = cells activated with tCD40L; black line = cells activated with either CD40 or LPS (in BL2-TLR4-NFκB-Luc cells); green line = cell toxicity, as measured by CellTiter-Glo.</p

A genome-wide association study of gout in people of European ancestry

Background/Purpose: Genome wide association studies (GWAS) have provided considerable insight into the molecular control of urate levels. However, less is known about the progression from asymptomatic hyperuricemia to gout. Our aim was to conduct a GWAS for gout in people of European ancestry using the largest number of cases of gout to date. Methods: This GWAS (7,431 cases and 105,631 controls) was comprised of three data sets: a mixed New Zealand (NZ), Eurogout, and Ardea Biosciences group (3,961 cases; 1,547 controls; genotyped using the Illumina CoreExome v24 array, 547,644 markers), a composite set from the Health Professionals Follow-Up (HPFS) and NursesÕ Health Studies (NHS) (1,038 cases; 1,095 controls; genotyped using the Illumina OmniExpress v12 array, 730,525 markers), and UK Biobank (2,432 cases; 102,989 controls; genotyped using an Affymetrix Axiom array, 820,967 markers). The UK Biobank genotypes were imputed to ~73.3M SNPs. Neither the NZ/Eurogout/Ardea nor NHS/HPFS genotype sets were imputed. Markers found within all three data sets (234,062) were identified and associated with gout (adjusted for sex and age), within each data set separately, using PLINK v1.9. An inverse-variance weighted meta-analysis of the results was then performed using the meta v4.4 package within R v3.2.3. The overall genome inflation factor was 0.90. Results: There were nine loci with experiment-wide significance (0.05/234,062; P < 2×10-7) for association with gout: ABCG2 (OR=1.77), SLC2A9 (OR=1.69), GCKR (OR=1.24), MLXIPL (OR=1.18), SLC17A1-A4 (OR=1.22), SLC16A9 (OR=1.18), SLC22A12 (OR=1.16), PDZK1 (OR=1.16), TRIM46 (OR=1.15). All nine of these loci have been previously associated with serum urate levels in genome-wide studies with the urate-increasing alleles also associated with increased risk of gout in this study. Conclusion: Our data emphasize the central importance of genetic involvement in serum urate levels, compared to the genetic involvement in MSU crystal formation, or the innate immune response, in determining gout.</p

Investigation of pleiotropic effects of RA-protecting <i>TYK2</i> variants using electronic medical records.

<p>We first tested association of the P1104A (A) and I684S (B) variants to 502 PheWAS phenotypes with frequency>1% in two independent EMR collections including 3,005 and 26,372 individuals of European ancestry, respectively. Pvalues of each PheWAS phenotype in meta-analysis of the two EMR collections are shown. We also tested association of the <i>TYK2</i> P1104A and I684S variants with low-density lipoproteins (LDL) levels (C), and white blood cell counts (WBC) (D). Effect sizes and confidence intervals in each EMR collection are shown. Pvalues from meta-analysis of the two EMR collections are indicated. Association results from SNPs previously reported to be associated with each quantitative trait (indicated by their respective rsIDs) are also shown.</p

Contribution of 3 independent <i>TYK2</i> protein-coding variants to protection from RA.

<p>(A) Three variants with MAF>0.5% predicted to be damaging and protecting from RA (P1104A, A928V and I684S) were identified using Immunochip data for 7,222 ACPA+ RA cases and 15,870 controls of European ancestry. (B) The three variants were genotyped in an independent dataset on the Exomechip (4,726 RA cases, 13,683 controls). (C) The three variants genotypes were also available from exon sequencing of <i>TYK2</i> in 1,118 RA cases, 1,118 matched controls of European ancestry. Frequencies of the independent haplotypes and odds ratios (OR) relative to the most frequent haplotype are shown. Minor alleles of the variants are highlighted in red. H, haplotypes; F, haplotype frequency; 1, P1104A; 2, A928V; 3, I684S.</p