Fresolimumab Treatment Decreases Biomarkers and Improves Clinical Symptoms in Systemic Sclerosis Patients

BACKGROUND. TGF-β has potent profibrotic activity in vitro and has long been implicated in systemic sclerosis (SSc), as expression of TGF-β–regulated genes is increased in the skin and lungs of patients with SSc. Therefore, inhibition of TGF-β may benefit these patients. METHODS. Patients with early, diffuse cutaneous SSc were enrolled in an open-label trial of fresolimumab, a high-affinity neutralizing antibody that targets all 3 TGF-β isoforms. Seven patients received two 1 mg/kg doses of fresolimumab, and eight patients received one 5 mg/kg dose of fresolimumab. Serial mid-forearm skin biopsies, performed before and after treatment, were analyzed for expression of the TGF-β–regulated biomarker genes thrombospondin-1 (THBS1) and cartilage oligomeric protein (COMP) and stained for myofibroblasts. Clinical skin disease was assessed using the modified Rodnan skin score (MRSS). RESULTS. In patient skin, THBS1 expression rapidly declined after fresolimumab treatment in both groups (P = 0.0313 at 7 weeks and P = 0.0156 at 3 weeks), and skin expression of COMP exhibited a strong downward trend in both groups. Clinical skin disease dramatically and rapidly decreased (P \u3c 0.001 at all time points). Expression levels of other TGF-β–regulated genes, including SERPINE1 and CTGF, declined (P = 0.049 and P = 0.012, respectively), and a 2-gene, longitudinal pharmacodynamic biomarker of SSc skin disease decreased after fresolimumab treatment (P = 0.0067). Dermal myofibroblast infiltration also declined in patient skin after fresolimumab (P \u3c 0.05). Baseline levels of THBS1 were predictive of reduced THBS1 expression and improved MRSS after fresolimumab treatment. CONCLUSION. The rapid inhibition of TGF-β–regulated gene expression in response to fresolimumab strongly implicates TGF-β in the pathogenesis of fibrosis in SSc. Parallel improvement in the MRSS indicates that fresolimumab rapidly reverses markers of skin fibrosis

miR-155 in the progression of lung fibrosis in systemic sclerosis

Background\ud MicroRNA (miRNA) control key elements of mRNA stability and likely contribute to the dysregulated lung gene expression observed in systemic sclerosis associated interstitial lung disease (SSc-ILD). We analyzed the miRNA gene expression of tissue and cells from patients with SSc-ILD. A chronic lung fibrotic murine model was used.\ud \ud Methods\ud RNA was isolated from lung tissue of 12 patients with SSc-ILD and 5 controls. High-resolution computed tomography (HRCT) was performed at baseline and 2–3 years after treatment. Lung fibroblasts and peripheral blood mononuclear cells (PBMC) were isolated from healthy controls and patients with SSc-ILD. miRNA and mRNA were analyzed by microarray, quantitative polymerase chain reaction, and/or Nanostring; pathway analysis was performed by DNA Intelligent Analysis (DIANA)-miRPath v2.0 software. Wild-type and miR-155 deficient (miR-155ko) mice were exposed to bleomycin.\ud \ud Results\ud Lung miRNA microarray data distinguished patients with SSc-ILD from healthy controls with 185 miRNA differentially expressed (q < 0.25). DIANA-miRPath revealed 57 Kyoto Encyclopedia of Genes and Genomes pathways related to the most dysregulated miRNA. miR-155 and miR-143 were strongly correlated with progression of the HRCT score. Lung fibroblasts only mildly expressed miR-155/miR-21 after several stimuli. miR-155 PBMC expression strongly correlated with lung function tests in SSc-ILD. miR-155ko mice developed milder lung fibrosis, survived longer, and weaker lung induction of several genes after bleomycin exposure compared to wild-type mice.\ud \ud Conclusions\ud miRNA are dysregulated in the lungs and PBMC of patients with SSc-ILD. Based on mRNA-miRNA interaction analysis and pathway tools, miRNA may play a role in the progression of the disease. Our findings suggest that targeting miR-155 might provide a novel therapeutic strategy for SSc-ILD

RA-MAP, molecular immunological landscapes in early rheumatoid arthritis and healthy vaccine recipients

Rheumatoid arthritis (RA) is a chronic inflammatory disorder with poorly defined aetiology characterised by synovial inflammation with variable disease severity and drug responsiveness. To investigate the peripheral blood immune cell landscape of early, drug naive RA, we performed comprehensive clinical and molecular profiling of 267 RA patients and 52 healthy vaccine recipients for up to 18 months to establish a high quality sample biobank including plasma, serum, peripheral blood cells, urine, genomic DNA, RNA from whole blood, lymphocyte and monocyte subsets. We have performed extensive multi-omic immune phenotyping, including genomic, metabolomic, proteomic, transcriptomic and autoantibody profiling. We anticipate that these detailed clinical and molecular data will serve as a fundamental resource offering insights into immune-mediated disease pathogenesis, progression and therapeutic response, ultimately contributing to the development and application of targeted therapies for RA.</p

Development and validation of a patient-reported outcome instrument for skin involvement in patients with systemic sclerosis

ObjectivesWe developed a patient-reported outcome (PRO) instrument to assess the skin-related quality of life in patients with systemic sclerosis (SSc).MethodsParticipants with SSc provided input on skin-related health effects through focus groups. We developed items for scleroderma skin PRO (SSPRO) to encompass these effects. Further consideration from cognitive interviews and an expert panel led to reduction and modification of items. A 22-item SSPRO was field tested. Psychometric analysis included test–retest reliability, internal consistency and exploratory factor analysis (EFA). Construct validity was assessed through correlation with other participant and physician-assessed measures.Results140 participants completed the SSPRO: mean age was 53.4 years, median disease duration was 5 years, 82.1% were female and 32.9% had diffuse cutaneous SSc. EFA supported four factors in SSPRO corresponding to hypothesised constructs: physical effects, physical limitations, emotional effects and social effects. Removal of 4/22 items resulted in acceptable goodness-of-fit statistics. Test–retest reliability (intraclass correlation coefficient=0.61–0.83) was moderate to high and internal consistency (Cronbach's α=0.89–0.96) was high. SSPRO correlated strongly with other participant-reported measures (r=0.59–0.88) suggesting construct validity, and less well with physician-assessed measures (r=0.31–0.40). SSPRO scores were significantly different for each level of participant-reported skin severity, and for limited versus diffuse cutaneous SSc.ConclusionsSSPRO has been developed with extensive patient input and demonstrates evidence for reliability and validity. It is complementary to existing measures of SSc skin involvement with emphasis on the patient's experience. Further research is needed to assess its sensitivity to change.</jats:sec

Brown fat determination and development from muscle precursor cells by novel action of bone morphogenetic protein 6.

Brown adipose tissue (BAT) plays a pivotal role in promoting energy expenditure by the virtue of uncoupling protein-1 (UCP-1) that differentiates BAT from its energy storing white adipose tissue (WAT) counterpart. The clinical implication of "classical" BAT (originates from Myf5 positive myoblastic lineage) or the "beige" fat (originates through trans-differentiation of WAT) activation in improving metabolic parameters is now becoming apparent. However, the inducers and endogenous molecular determinants that govern the lineage commitment and differentiation of classical BAT remain obscure. We report here that in the absence of any forced gene expression, stimulation with bone morphogenetic protein 6 (BMP6) induces brown fat differentiation from skeletal muscle precursor cells of murine and human origins. Through a comprehensive transcriptional profiling approach, we have discovered that two days of BMP6 stimulation in C2C12 myoblast cells is sufficient to induce genes characteristic of brown preadipocytes. This developmental switch is modulated in part by newly identified regulators, Optineurin (Optn) and Cyclooxygenase-2 (Cox2). Furthermore, pathway analyses using the Causal Reasoning Engine (CRE) identified additional potential causal drivers of this BMP6 induced commitment switch. Subsequent analyses to decipher key pathway that facilitates terminal differentiation of these BMP6 primed cells identified a key role for Insulin Like Growth Factor-1 Receptor (IGF-1R). Collectively these data highlight a therapeutically innovative role for BMP6 by providing a means to enhance the amount of myogenic lineage derived brown fat

Adipocytes derived from C2C12 cells by BMP6 action exhibit bioenergetics phenotype characteristic of brown adipocytes.

<p>Oxygen consumption rate (OCR) measurements for (A) basal respiration, (B) ATP turn over, (C) proton leak and (D) fatty acid oxidation using a Seahorse XF24 extracellular flux analyzer following sequential administration of oligomycin, FCCP and rotenone. Results in (A) are plotted relative to unstimulated condition set to 100. Data in (B) and (C) are expressed as percent of basal respiration. Results represent cumulative analyses of multiple independent biological replicates from three independent experiments (n≥6 for each condition per experiment). (D) Fatty acid (palmitate) oxidation capacity is represented as increased OCR in response to palmitate-BSA complex compared to BSA only as control. Results represent cumulative analyses of multiple independent biological replicates from three independent experiments (n≥9 for palmitate-BSA and ≥9 for BSA for each condition, mean ± SD). <i>*p<0.05</i> versus no BMP.</p

BMP6 induces a strong brown fat gene program in C2C12 cells without inducing <i>Prdm16.</i>

<p>(<b>A</b>) Q-PCR analyses of BAT thermogenic markers (<i>Ucp-1</i>, <i>Dio2</i> and <i>Pgc-1α</i>) and BAT specific markers (<i>Cidea</i> and <i>Elovl3</i>) in BMP6 or BMP7 (250 ng/mL) pretreated cells followed by adipogenic differentiation and stimulation with or without 10 μM forskolin for 4 hours. (<b>B</b>) Parallel analyses of BAT thermogenic markers in BMP8 (250 ng/mL) pretreated cells. The expression in untreated cells (no BMP pretreatment and no forskolin stimulation: no BMP-no F) was set to 1, and results represent triplicate analyses of three independent biological replicates (mean ± SD), <i>*p<0.05</i> versus no BMP-F or no BMP, Student's <i>t</i> test. Similar results were obtained in at least two independent analyses. (<b>C</b>) Q-PCR generated Ct values for <i>Prdm16</i> and <i>Gapdh</i> transcripts at the end of differentiation cascade following BMP6 or BMP7 stimulation. (<b>D</b>) Ct values for <i>Prdm16</i> and <i>Gapdh</i> in relevant phases of differentiation landscape following BMP6 stimulation. Each dot per phase represents an independent biological replicate. For all the Q-PCR analyses, <i>Gapdh</i> served as the endogenous control. (<b>E</b>) Heat map depiction and unbiased clustering analyses of 1718 significantly changing qualifiers (<i>p≤0.05, fold change≥1.5</i>) in C2C12 cells stimulated with or without BMP6 for two days followed by differentiation protocol outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092608#pone-0092608-g001" target="_blank">Figure 1A</a>. Key brown fat and pan-adipogenic markers: <i>Fabp4, Adiponectin, Fabp5, Monoglyceride lipase, Dio2, Lipoprotein lipase (Lpl), Perilipin 4, Pparγ, and CD36,</i> and myogenic markers<i>: Calsequestrin 2 (Casq2), Myozenin 1 (Myoz1), Tropomyosin 3 (Tpm3), Myosin binding protein C-fast type (Mybpc2) and Myosin regulatory light chain 2 (Myl2)</i> are highlighted in the heat map. Each gene is represented by a single row and each sample in three independent biological replicates for each time point, by a column. Two distinct clusters indicate genes induced (red) and repressed (green), with representative induced and repressed genes highlighted in red and green boxes respectively.</p