Profiling microRNAs in individuals at risk of progression to rheumatoid arthritis

Background: Individuals at risk of rheumatoid arthritis (RA) demonstrate systemic autoimmunity in the form of anti-citrullinated peptide antibodies (ACPA). MicroRNAs (miRNAs) are implicated in established RA. This study aimed to (1) compare miRNA expression between healthy individuals and those at risk of and those that develop RA, (2) evaluate the change in expression of miRNA from "at-risk" to early RA and (3) explore whether these miRNAs could inform a signature predictive of progression from "at-risk" to RA. Methods: We performed global profiling of 754 miRNAs per patient on a matched serum sample cohort of 12 anti-cyclic citrullinated peptide (CCP) + "at-risk" individuals that progressed to RA. Each individual had a serum sample from baseline and at time of detection of synovitis, forming the matched element. Healthy controls were also studied. miRNAs with a fold difference/fold change of four in expression level met our primary criterion for selection as candidate miRNAs. Validation of the miRNAs of interest was conducted using custom miRNA array cards on matched samples (baseline and follow up) in 24 CCP+ individuals; 12 RA progressors and 12 RA non-progressors. Results: We report on the first study to use matched serum samples and a comprehensive miRNA array approach to identify in particular, three miRNAs (miR-22, miR-486-3p, and miR-382) associated with progression from systemic autoimmunity to RA inflammation. MiR-22 demonstrated significant fold difference between progressors and non-progressors indicating a potential biomarker role for at-risk individuals. Conclusions: This first study using a cohort with matched serum samples provides important mechanistic insights in the transition from systemic autoimmunity to inflammatory disease for future investigation, and with further evaluation, might also serve as a predictive biomarker

Heterarchy of Transcription Factors Driving Basal and Luminal Cell Phenotypes in Human Urothelium

Cell differentiation is effected by complex networks of transcription factors that co-ordinate re-organisation of the chromatin landscape. The hierarchies of these relationships can be difficult to dissect. During in vitro differentiation of normal human uro-epithelial cells, formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) and RNA-seq were used to identify alterations in chromatin accessibility and gene expression changes following activation of the nuclear receptor PPARG as a differentiation-initiating event. Regions of chromatin identified by FAIRE-seq as having altered accessibility during differentiation were found to be enriched with sequence-specific binding motifs for transcription factors predicted to be involved in driving basal and differentiated urothelial cell phenotypes, including FOXA1, P63, GRHL2, CTCF and GATA3. In addition, co-occurrence of GATA3 motifs was observed within sub-sets of differentiation-specific peaks containing P63 or FOXA1 after induction of differentiation. Changes in abundance of GRHL2, GATA3, and P63 were observed in immunoblots of chromatin-enriched extracts. Transient siRNA knockdown of P63 revealed that P63 favoured a basal-like phenotype by inhibiting differentiation and promoting expression of basal marker genes. GATA3 siRNA prevented differentiation-associated downregulation of P63 protein and transcript, and demonstrated positive feedback of GATA3 on PPARG transcript, but showed no effect on FOXA1 transcript or protein expression. This approach indicates that as a transcriptionally-regulated programme, urothelial differentiation operates as a heterarchy wherein GATA3 is able to co-operate with FOXA1 to drive expression of luminal marker genes, but that P63 has potential to transrepress expression of the same genes

Heterochromatin protein 1 gamma and IiB kinase alpha interdependence during tumour necrosis factor gene transcription elongation in activated macrophages

IiB kinase a (IKKa) is part of the cytoplasmic IKK complex regulating nuclear factor-{kappa}B (NF-iB) release and translocation into the nucleus in response to pro-inflammatory signals. IKKa can also be recruited directly to the promoter of NF-iB-dependent genes by NF-iB where it phosphorylates histone H3 at serine 10, triggering recruitment of the bromodomain-containing protein 4 and the positive transcription elongation factor b. Herein, we report that IKKa travels with the elongating form of ribonucleic acid polymerase II together with heterochromatin protein 1 gamma (HP1c) at NF-iBdependent genes in activated macrophages. IKKa binds to and phosphorylates HP1c, which in turn controls IKKa binding to chromatin and phosphorylation of the histone variant H3.3 at serine 31 within transcribing regions. Downstream of transcription end sites, IKKa accumulates with its inhibitor the CUE-domain containing protein 2, suggesting a link between IKKa inactivation and transcription termination

Effect of tocilizumab therapy on il-6 signalling pathway in early rheumatoid arthritis patients

Background: We previously reported increased constitutive but unchanged induced CD4+ p-STAT3 in established RA versus healthy control (HC).¹ This study aims to evaluate in early RA (ERA) (i) cell-subset constitutive and Cis (IL-6) and Trans (IL-6/sIL6R)-induced IL-6 signalling (ii) effect of tocilizumab (TCZ; +/-MTX) (iii) for an association between baseline phosphorylation intensities and TCZ (+/- MTX) response. Materials and methods: Multiparameter phosphoflow cytometry was performed on 20 treatment-naïve, ERA patients randomised to TCZ monotherapy or TCZ-MTX to determine STAT3, Akt and Erk phosphorylation intensities. PBMCs were isolated and cryopreserved at baseline, and weeks 24 and 48 post-TCZ. HC samples (n=10) were also obtained. PBMCs were unmodulated, stimulated with IL-6 or soluble IL-6 receptor (sIL-6R) and median fluorescence intensities (MFI) were measured. Results Baseline data, HC and ERA: Constitutive CD4+ p-STAT3 was numerically higher in ERA compared to HC, with non-significant increase following stimulation in both groups. There was no significant difference in constitutive monocyte p-STAT3 in HC vs ERA. Stimulation did not induce further increase in monocyte p-STAT3 in ERA. Constitutive CD4+ p-Akt was significantly higher in ERA versus HC (p=0.01), with no change post-stimulation. Pre/Post TCZ in ERA: Constitutive CD4+ pSTAT3 expression did not alter following TCZ exposure. Statistically significant decreases in IL-6 stimulated CD4+ pSTAT3 expression from baseline to weeks 24 and 48 were observed (p=0.005 and 0.01 respectively); and similarly using IL-6/sIL-6R at week 48 (p=0.01). Constitutive monocyte p-STAT3 expression appeared to increase at week 24 compared to baseline (p=0.03); similar observation following stimulation with IL-6/sIL-6R at both weeks 24 and 48 was observed (p=0.01). TCZ led to non-significant decrease in constitutive and IL6-stimulated CD4+ p-Akt expression weeks 24 and 48. TCZ otherwise had no significant effect on p-Akt and p-Erk in other studied cell subsets. Conclusions: TCZ reduced induced IL-6 signalling via CD4+ STAT3. This preliminary analysis also suggests importance of monocyte subset and relevance of Akt in IL-6 signalling. Complete analysis of these data will clarify the relative roles and impact of IL-6R targeting further

TLR dependent XBP-1 activation induces an autocrine loop in rheumatoid arthritis synoviocytes

X-box binding protein 1 (XBP1) is a central regulator of the endoplasmic reticulum (ER) stress response. It is induced via activation of the IRE1 stress sensor as part of the unfolded protein response (UPR) and has been implicated in several diseases processes. XBP1 can also be activated in direct response to Toll-like receptor (TLR) ligation independently of the UPR but the pathogenic significance of this mode of XBP1 activation is not well understood. Here we show that TLR-dependent XBP1 activation is operative in the synovial fibroblasts (SF) of patients with active rheumatoid arthritis (RA). We investigated the expression of ER stress response genes in patients with active RA and also in patients in remission. The active (spliced) form of (s)XBP1 was significantly overexpressed in the active RA group compared to healthy controls and patients in remission. Paradoxically, expression of nine other ER stress response genes was reduced in active RA compared to patients in remission, suggestive of a UPR-independent process. However, sXBP1 was induced in SF by TLR4 and TLR2 stimulation, resulting in sXBP1-dependent interleukin-6 and tumour necrosis factor (TNF) production. We also show that TNF itself induces sXBP1 in SF, thus generating a potential feedback loop for sustained SF activation. These data confirm the first link between TLR-dependent XBP1 activation and human inflammatory disease. sXBP1 appears to play a central role in this process by providing a convergence point for two different stimuli to maintain activation of SF

Additional file 10: of Profiling microRNAs in individuals at risk of progression to rheumatoid arthritis

Network of the predicted targets of miR-382. (DOCX 214 kb

Additional file 4: of Profiling microRNAs in individuals at risk of progression to rheumatoid arthritis

List of miRNAs of interest with age adjustment in the validation phase. (DOCX 119 kb

Additional file 3: of Profiling microRNAs in individuals at risk of progression to rheumatoid arthritis

Serum miRNAs miR-146a and miR-155 expression levels in the pilot phase. (DOCX 19324 kb