Background/Purpose Osteoarthritis (OA) is the leading cause of chronic disability affecting 40% of individuals over the age of 70 and costing $128 billion annually in the US alone. Little is known regarding changes in gene expression that occur regionally within these affected joints. Herein, we perform RNA-seq analysis of eroded and intact cartilage from human OA, and correlate transcript levels with histopathologic disease severity. Methods Six femoral heads were obtained at the time of hip arthroplasty for primary OA. Articular cartilage tissue was dissected from grossly affected and grossly normal areas from the same joints, flash frozen in liquid nitrogen, and RNA was extracted. Tissue samples from grossly affected and normal joint regions were histologically examined for OA severity using Mankin scoring. Following confirmation of RNA quality (RIN value ≥6), samples were sequenced with the Illumina TruSeq system on a MiSeq sequencer. Raw data were analysed and mapped using the GeneSifter software package. Genes with GeneSifter quality score <1.0 were excluded. For categorical analysis, EdgeR p-values <0.05 with Benjamini-Hochberg q ≤ 0.1 and expression ratios ≤0.83 or ≥ 1.2 between affected and normal tissues were considered significant. For correlations with histologic score, Pearson’s r > 0.75 or <-0.75 with p ≤ 0.05 were considered significant. Gene ontology and pathway analysis was performed using Ingenuity IPA and DAVID. Results Categorical analysis identified 43 overexpressed and 313 underexpressed genes in eroded compared to intact cartilage. Both over- and underexpressed genes were overrepresented in the fibroblastic growth factor (FGF) signalling pathway (p = 0.004). FGFR2 demonstrated aneroded to intact cartilage expression ratio = 0.46, was highly inversely correlated with OA histologic score severity (r = 0.92), and is hypermethylated in eroded OA cartilage. The WNT pathway genes, WNT11 (ratio 0.27) and WNT9A (ratio 0.45), and STAT3 pathway was also overrepresented, including both over- and underexpressed genes (p = 0.001). A top predicted upstream regulator in differentially expressed genes was mir-9 (p = 0.005), known to be associated with metalloproteinase production. Further, we identified 1576 genes positively correlated with histopathologic score. Among these, the NFAT pathway was highly overrepresented including both positively and inversely correlated genes (24, p = 0.002). The RIG-I innate immunity pathway was also overrepresented among inversely correlated genes (p = 0.008), as were several genes related to chromatin remodelling (overall p = 0.009: HDAC1, r = -0.89, MECP2 r = -0.78, RBBP4 r = -0.82, SAP130 r = -0.81, SIN3A r = -0.80). Conclusions Using RNA-seq we detected significant changes in gene expression in eroded compared to intact OA cartilage, as well as expression changes correlated with histologic disease progression in OA. Our data strongly suggest involvement of several signalling pathways, many of which are potential therapeutic targets for OA. This work reinforces ...

Objectives: Binding of the bromodomain and extra-terminal domain proteins (BETs) to acetylated histone residues is critical for gene transcription in many fibrotic diseases. This study sought to determine the anti-fibrotic efficacy and potential mechanisms of BET inhibition in scleroderma (SSc). Methods: Dermal fibroblasts were isolated from biopsies from healthy subjects or patients with diffuse cutaneous (dc)SSc. Fibroblasts were treated with pan BET inhibitor JQ1, BRD2 inhibitor BIC1, or BRD4 inhibitors AZD5153 or ARV825. Knockdown of BETs was achieved by siRNA transfection. The in vivo anti-fibrotic efficacy of JQ1 was determined in a bleomycin-induced skin fibrosis mouse model. T-test or ANOVA were used to compare differences between groups, and a p-value of <0.05 was considered significant. Results: BET inhibitor JQ1 dose-dependently downregulated pro-fibrotic genes in dcSSc fibroblasts, and inhibited cell migration and gel contraction. It suppressed proliferation by inducing cell cycle arrest. The anti-fibrotic effects of JQ1 were also observed in TGFβ-treated normal fibroblasts. JQ1 prevented bleomycin-induced skin fibrosis in mice. The anti-fibrotic effect of JQ1 was mediated by inhibition of BRD4, as specific blockade or knockdown of BRD4 led to downregulation of fibrotic markers and inhibition of myofibroblast properties, while inhibition or knockdown of BRD2 or BRD3 had minimal effects in dcSSc fibroblasts. Conclusions: BET inhibition showed promising anti-fibrotic effects in SSc both in vitro and in vivo. Specifically, we showed that BRD4 plays a critical role in SSc fibrosis and that targeting BRD4 might be a novel therapeutic option for this disease.

Objectives: Caffeine is a widely consumed pharmacologically active product. In the present study, we focused on characterizing immunomodulatory effects of caffeine on peripheral blood mononuclear cells (PMBCs). Methods:The effect of caffeine on gene expression profiles was initially evaluated using RNA sequencing data. Validation experiments were performed to confirm the results and examine dose-dependent effects of caffeine on PBMCs from healthy subjects. Gene expression levels were measured by real-time quantitative PCR, and cytokine production was determined using a multiplex cytokine assay.Results: Caffeine at high doses showed a robust downregulatory effect of immunerelated genes in PBMCs. Functional annotation analysis of downregulated genes revealed significant enrichment in cytokine activity and in genes related to several autoimmune diseases including lupus and rheumatoid arthritis. Dose-dependent validation experiments showed significant downregulation at the mRNA levels of key inflammatory genes including STAT1, TNF, and PPARG. TNF and PPARG were suppressed even with the lowest caffeine dose tested, which corresponds to the serum concentration of caffeine after administration of one cup of coffee. Cytokine levels of IL-8, MIP-1β, IL-6, IFN-γ, GM-CSF, TNF, IL-2, IL-4, MCP-1, and IL-10 were decreased significantly with caffeine treatment. Conclusion:Our findings indicate potential downregulatory effects of caffeine on key inflammatory genes and cytokines, which play important role in autoimmunity.Further studies exploring therapeutic or disease-modulating potential of caffeine in autoimmune diseases and exploring the mechanisms involved are warranted. peer-reviewed)

BackgroundSegmental duplication affecting the FCGR locus on chromosome 1 has led to ambiguous mapping of underlying genetic variants. Consequently the region is misrepresented in SNP databases, has been untested by genome wide association studies and dismissed as a potential contributor to genetic predisposition. Several large-scale studies identified genetic markers close to FCGR2A with divergent, yet reproducible associations with a number of autoimmune and inflammatory diseases. Our objective was to define the causal variants associated with FcγRIIa in disease susceptibility.MethodsGene specific resequencing of FCGR2A was performed in a panel of 32 controls of mixed ethnicity. Fine scale linkage disequilibrium was used to infer trans-segmental duplication marker relationships, allowing reanalysis of Immunochip data from several autoimmune and inflammatory disease studies. Immunochip data for the FCGR2A locus from UK and Spanish RA, Spanish systemic sclerosis, Caucasian ankylosing spondylitis, UK and Irish psoriatic arthritis, European systemic lupus erythematosus, UK myositis, UK and Spanish giant cell arteritis and US/Turkish Takayasu arteritis cohorts were reanalysed. FcγRIIa ectodomain proteins of each allotype were synthesised and their IgG subclass binding preferences and biophysical properties were interrogated using surface plasmon resonance and SEC-MALLS.ResultsRe-analysis of Immunochip data identified two groups of associated SNPs stratified by minor allele frequency, tagging Q27W and H131R substitutions. Haplotypes of these two risk markers showed different susceptibility associations with inflammatory diseases compared to the common 27Q-131R haplotype. Both UK and Spanish RA (OR (95% CI) 1.17 (1.07–1.27) p = 0.0003 and 1.24 (1.05–1.47) p = 0.011 respectively) and US Takayasu arteritis (OR 1.78 (1.21–2.63) p = 0.0037 were associated with the 27W-131H haplotype. The 27Q-131H haplotype predisposed to ankylosing spondylitis in Europeans (OR 1.15 (1.10–1.20) p = 2.04 × 10–11), but was protective from SLE (OR 0.79 (0.74–0.84) p = 1.3 × 10–13) and myositis (OR 0.83 (0.72–0.95) p = 0.0083). Using biophysical techniques these nonsynonymous coding SNPs altered  the affinity and binding preferences of the receptor for IgG subclasses and influenced receptor oligomerisation.ConclusionSegmental duplication and structural variation confounded iChip SNP analyses, however FCGR2A specific resequencing allowed analysis of trans-segmental duplication markers. We confirmed two independent genetic risk factors at this locus. The functional impact and potential pathogenic role for FCGR2A in autoimmune/inflammatory disease is discussed.

We have previously revealed a genetic association between Takayasu arteritis and a non-coding genetic variant in an enhancer region within IL6 (rs2069837 A/G). The risk allele in this variant (allele A) has a protective effect against chronic viral infection and cancer. Using a combination of experimental and bioinformatics tools, we identified the monocyte/macrophage anti-inflammatory gene GPNMB, ~520kb away, as a target gene regulated by rs2069837. We revealed preferential recruitment of myocyte enhancer factor 2-histone deacetylase (MEF2-HDAC) repressive complex to the Takayasu arteritis risk allele. Further, we demonstrated suppression of GPNMB expression in monocyte-derived macrophages from healthy individuals with the AA compared to AG genotype, which was reversed by histone deacetylase inhibition. Our data suggest that the A allele in rs2069837 represses the expression of GPNMB by recruiting MEF2-HDAC complex, enabled through a long-range intra-chromatin looping mediated by CTCF. Suppression of this anti-inflammatory gene might mediate increased susceptibility in Takayasu arteritis and enhance protective immune responses in chronic infection and cancer. Our data highlight long-range chromatin interactions in functional genomic studies.