Tobias Messemaker

The Effects of Low Levels of Dystrophin on Mouse Muscle Function and Pathology

Duchenne muscular dystrophy (DMD) is a severe progressive muscular disorder caused by reading frame disrupting mutations in the DMD gene, preventing the synthesis of functional dystrophin. As dystrophin provides muscle fiber stability during contractions, dystrophin negative fibers are prone to exercise-induced damage. Upon exhaustion of the regenerative capacity, fibers will be replaced by fibrotic and fat tissue resulting in a progressive loss of function eventually leading to death in the early thirties. With several promising approaches for the treatment of DMD aiming at dystrophin restoration in clinical trials, there is an increasing need to determine more precisely which dystrophin levels are sufficient to restore muscle fiber integrity, protect against muscle damage and improve muscle function. To address this we generated a new mouse model (mdx-Xist Δhs) with varying, low dystrophin levels (3–47%, mean 22.7%, stdev 12.1, n = 24) due to skewed X-inactivation. Longitudinal sections revealed that within individual fibers, some nuclei did and some did not express dystrophin, resulting in a random, mosaic pattern of dystrophin expression within fibers. Mdx-Xist Δhs, mdx and wild type females underwent a 12 week functional test regime consisting of different tests to assess muscle function at base line, or after chronic treadmill running exercise. Overall, mdx-Xist Δhs mice with 3–14% dystrophin outperformed mdx mice in the functional tests. Improved histopathology was observed in mice with 15–29% dystrophin and these levels also resulted in normalized expression of pro-inflammatory biomarker genes, while for other parameters >30% of dystrophin was needed. Chronic exercise clearly worsened pathology, which needed dystrophin levels >20% for protection. Based on these findings, we conclude that while even dystrophin levels below 15% can improve pathology and performance, levels of >20% are needed to fully protect muscle fibers from exercise-induced damage.

Immunogenetics of rheumatoid arthritis: Understanding functional implications.

The last decade has seen a dramatic technological revolution. The characterisation of the majority of the common variations in our genetic code in 2003 precipitated the discovery of the genetic risk factors predisposing to Rheumatoid Arthritis development and progression. Prior to 2007, only a handful of genetic risk factors had been identified, HLA, PTPN22 and CTLA4. Since then, over 100 genetic risk loci have been described, with the prediction that an ever-increasing number of risk alleles with consistently decreasing effect sizes will be discovered in the years to come. Each risk locus harbours multiple candidate genes and the proof of causality of each of these candidates is as yet unknown. An enrichment of these RA-associated genes is found in three pathways: T-cell receptor signalling, JAK-STAT signalling and the NF-κB signalling cascade, and currently drugs targeting these pathways are available for the treatment of RA. However, the role that RA-associated genes have in these pathways and how they contribute to disease is not always clear. Major efforts in understanding the contribution of genetic risk factors are currently under way with studies querying the role of genetic variation in gene expression of coding and non-coding genes, epigenetic marks and other regulatory mechanisms yielding ever more valuable insights into mechanisms of disease. Recent work has suggested a possible enrichment of non-coding RNAs as well as super-enhancers in RA genetic loci indicating possible new insights into disease mechanism. This review brings together these emerging genetic data with an emphasis on the immunogenetic links these findings have provided and what we expect the future will bring.

Exome Sequencing Identifies A Branch Point Variant in Aarskog–Scott Syndrome

Aarskog–Scott syndrome (ASS) is a rare disorder with characteristic facial, skeletal, and genital abnormalities. Mutations in the FGD1 gene (Xp11.21) are responsible for ASS. However, mutation detection rates are low. Here, we report a family with ASS where conventional Sanger sequencing failed to detect a pathogenic change in FGD1. To identify the causative gene, we performed whole‐exome sequencing in two patients. An initial analysis did not reveal a likely candidate gene. After relaxing our filtering criteria, accepting larger intronic segments, we unexpectedly identified a branch point (BP) variant in FGD1. Analysis of patient‐derived RNA showed complete skipping of exon 13, leading to premature translation termination. The BP variant detected is one of very few reported so far proven to affect splicing. Our results show that besides digging deeper to reveal nonobvious variants, isolation and analysis of RNA provides a valuable but under‐appreciated tool to resolve cases with unknown genetic defects.

Allele-specific repression of Sox2 through the long non-coding RNA Sox2ot

The transcription factor Sox2 controls the fate of pluripotent stem cells and neural stem cells. This gatekeeper function requires well-regulated Sox2 levels. We postulated that Sox2 regulation is partially controlled by the Sox2 overlapping long non-coding RNA (lncRNA) gene Sox2ot. Here we show that the RNA levels of Sox2ot and Sox2 are inversely correlated during neural differentiation of mouse embryonic stem cells (ESCs). Through allele-specific enhanced transcription of Sox2ot in mouse Sox2eGFP knockin ESCs we demonstrate that increased Sox2ot transcriptional activity reduces Sox2 RNA levels in an allele-specific manner. Enhanced Sox2ot transcription, yielding lower Sox2 RNA levels, correlates with a decreased chromatin interaction of the upstream regulatory sequence of Sox2 and the ESC-specific Sox2 super enhancer. Our study indicates that, in addition to previously reported in trans mechanisms, Sox2ot can regulate Sox2 by an allele-specific mechanism, in particular during development.

Inflammatory genes TNFα and IL6 display no signs of increased H3K4me3 in circulating monocytes from untreated rheumatoid arthritis patients

Innate immune cells, such as monocytes, can adopt a long-lasting pro-inflammatory phenotype, a phenomenon called ‘trained immunity’. In trained immunity, increased cytokine levels of genes, like interleukin (IL)-6 and tumor necrosis factor (TNF)-α, are observed, which are associated with increased histone 3 lysine 4 trimethylation (H3K4me3) in the promoter region. As systemic IL6 and TNFα levels are increased in rheumatoid arthritis (RA) patients and monocytes are known to be the primary producers of TNFα and IL6, we hypothesized that ‘trained immunity’ signals may be observed at these genes in monocytes from RA patients. CD14+ monocytes were isolated from untreated RA patients and paired age-matched healthy controls. H3K4me3, mRNA, protein and serum levels of IL6 and TNFα were evaluated by chromatin immunoprecipitation, reverse-transcription quantitative PCR and enzyme-linked immunosorbent assays. Despite elevated serum levels of TNFα and IL6 in the tested RA patients (P<0.05), ex vivo isolated monocytes displayed similar H3K4me3 levels to healthy controls in the promoter region of TNFα and IL6. Concordantly, mRNA and protein levels of IL6 and TNFα were similar before and after lipopolysaccharide stimulation between patients and controls. Together, with the current number of individuals tested we have not detected enhanced trained immunity signals in circulating monocytes from untreated RA patients, despite increased IL6 and TNFα serum levels.

Comment on “Functional Analysis of a Complement Polymorphism (rs17611) Associated with Rheumatoid Arthritis”

In their published article, Giles et al. ([1][1]) explored functional consequences of rs17611 in the TRAF1-C5 region and propose a mechanism for its contribution to rheumatoid arthritis (RA) pathology, as this SNP would associate with RA. To substantiate the claim that the SNP associates with RA,

Mast cells in early rheumatoid arthritis associate with disease severity and support B cell autoantibody production

Objectives Mast cells (MCs) are involved in the pathogenesis of rheumatoid arthritis (RA). However, their contribution remains controversial. To establish their role in RA, we analysed their presence in the synovium of treatment-naïve patients with early RA and their association and functional relationship with histological features of synovitis. Methods Synovial tissue was obtained by ultrasound-guided biopsy from treatment-naïve patients with early RA (n=99). Immune cells (CD3/CD20/CD138/CD68) and their relationship with CD117+MCs in synovial tissue were analysed by immunohistochemistry (IHC) and immunofluorescence (IF). The functional involvement of MCs in ectopic lymphoid structures (ELS) was investigated in vitro, by coculturing MCs with naïve B cells and anticitrullinated protein antibodies (ACPA)-producing B cell clones, and in vivo in interleukin-27 receptor alpha (IL27ra)-deficient and control mice during antigen-induced arthritis (AIA). Results High synovial MC counts are associated with local and systemic inflammation, autoantibody positivity and high disease activity. IHC/IF showed that MCs reside at the outer border of lymphoid aggregates. Furthermore, human MCs promote the activation and differentiation of naïve B cells and induce the production of ACPA, mainly via contact-dependent interactions. In AIA, synovial MC numbers increase in IL27ra deficient mice, in association with ELS and worse disease activity. Conclusions Synovial MCs identify early RA patients with a severe clinical form of synovitis characterised by the presence of ELS.

FRI0356 Antisense long noncoding rnas are deregulated in skin tissue of ssc patients

Background Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of skin and multiple organs of which pathogenesis is poorly understood. Here we studied differentially expressed coding and non-coding genes in relation to SSc pathogenesis with a specific focus on antisense non-coding RNAs. Objectives Here we studied differentially expressed coding and non-coding genes in relation to SSc pathogenesis with a specific focus on antisense non-coding RNAs. Methods Skin biopsy-derived RNAs from fourteen early SSc patients and six healthy individuals were sequenced with ion-torrent and analysed using DEseq2. Protein-coding and non-coding genes annotated in GENCODEV7 were analysed. Significant long non-coding RNAs were independently replicated in a Northern American dataset. Results 4901 genes with a fold change >1.5 and a false discovery rate of less than 5% were detected in patients versus controls. Upregulated coding genes clustered in immunological, cell adhesion and keratin-related processes as previously found by microarray studies. Interestingly, 676 deregulated non-coding genes were detected, 257 of which were classified as antisense genes. 42% of these antisense genes had a concurrent deregulated sense gene. The majority of the sense-antisense genes had a similar effect sizes in an independent North American dataset with three genes (OTUD6B-AS1, CTBP1-AS2 and HMGN3-AS1) exceeding the study-wide Bonferroni-corrected ρ-value (PBonf<0.0024, Pcombined=1.6x10-9, 1.7x10-6, 2.6xx10-6, respectively). Intriguingly, the correlation of sense-antisense gene pairs deregulated in SSc is stronger than sense-antisense gene pairs not deregulated in SSc (p<0.001). Conclusions For the first time we highlight that together with coding genes, (antisense) long noncoding RNAs are deregulated in skin tissue of SSc patients suggesting a novel class of genes involved in pathogenesis of SSc. Disclosure of Interest None declared

A8.09 Trained immunity in monocytes from rheumatoid arthritis patients and healthy individuals

Background and objective Rheumatoid Arthritis (RA) is a heterogeneous disease affecting approximately 1–2% of the European population. Development of RA is affected by both genetic and environmental triggers. In the present study we hypothesised that these environmental triggers might induce changes in the epigenome of immune cells. A concept that describes such a mechanism is known as ‘trained immunity’, which shows that isolated monocytes previously exposed to micro-organisms display elevated H3K4me3 on the promotor regions of immune genes resulting in enhanced RNA- and protein levels of TNFa and IL6 upon re-stimulation with inflammatory triggers. The phenomenon that innate cells triggered by past insults more readily react to new non-specific stimuli could contribute to the onset of auto-immunity. We wished to test whether the innate immune system of RA patients displays signs of “trained immunity” by analysing immune responsiveness and epigenetic changes of CD14+ monocytes isolated from both untreated RA patients and age-matched healthy controls. Methods CD14+ monocytes were isolated from both untreated RA patients and age-matched healthy controls. Secreted TNFa and IL6 levels were measured using ELISA in the supernatant of naïve and LPS-stimulated monocytes from both RA patients and non-RA controls. RNA levels of TNFa and IL6 in naïve and LPS-stimulated monocytes from both groups were examined using RT-qPCR. Results Both RNA and protein levels of TNFa and IL6 were low in unstimulated monocytes isolated from RA patients and healthy controls but no large differences were observed. After LPS-stimulation, the relative RNA levels (expressed in fold change relative to household gene) increased but were similar between patients and controls for TNFa (RA: 1.7 ± 0,4, non-RA: 1.8 ± 0.9) and IL6 (RA: 2.9 ± 1,3, non-RA: 3.3 ± 2.6). Concordantly, no large differences were detected for secreted cytokine levels, although secreted TNFa (RA: 4.3ng/ml ± 2.9, non-RA: 2.8ng/ml ± 2.6) and IL6 (RA: 67.2mg/ml ± 14.6, non-RA: 50.8mg/ml ± 17.3) levels seem slightly elevated in RA monocytes. Conclusions In general, we could not detect large differences on RNA level or protein level of TNFa and IL6 between RA and non-RA patients. Therefore, we did not find evidence that the concept of trained immunity may contribute to the development of RA.

FRI0247 The Involvement of The Long Noncoding H19x in tGFβ Signaling and Its Profibrotic Effects in Systemic Sclerosis and Other Fibrotic Diseases

Background Long noncoding RNAs (lncRNAs) are an emerging class of noncoding transcripts involved in the regulation of gene expression in health and disease. We have recently identified a novel lncRNA, H19X, which is strictly regulated by TGFβ in a dose and time dependent manner. Objectives To detail the differential expression of H19X in SSc and other fibrotic diseases and to characterize its function in fibrotic diseases. Methods Skin and lung biopsies from patients with SSc, idiopathic lung fibrosis (IPF), and healthy controls (HC) were obtained from cohorts at four different expert centers. Expression of H19X was analyzed by RNA Sequencing Ilumina HiSeq2000 and real-time PCR respectively. H19X was silenced in skin fibroblasts using locked nucleic acid antisense oligonucleotides (LNA GapmeRs), followed by qPCR analyses, immunofluorescence staining, SIRCOL assay, contraction assay and Western blot. In situ hybridization of H19X on SSc dermal fibroblast was performed using Stellaris FISH probes. Results H19X expression was consistently upregulated across all four cohorts (SSc n=34, HC n=26). Notably, the upregulation was also consistent across different subsets of patients. H19X showed a similar expression profile in clinically non-involved skin compared to established fibrotic skin. Moreover, expression of H19X was also significantly increased in SSc interstitial lung disease patients versus HC (n=11 each, p<0.05). A significant H19X overexpression was also detected in IPF samples indicating a broader role of H19X in fibrotic diseases (n=11 each, p<0.05). The knockdown of H19X by GapmeRs in skin fibroblasts led to a strong downregulation of collagen 1, fibronectin and αSMA mRNAs (n=5, p<0.05). Furthermore, H19X knockdown followed by SIRCOL assay and Western blot analysis confirmed the importance of this lncRNA in the regulation of extracellular matrix components such as collagens and fibronectin. Additionally, silencing of H19X significantly impaired αSMA fiber formation, stress fiber formation and cell contractility showing an important role of H19X in the development of the myofibroblast phenotype. Because the H19X gene hosts the sequence for two microRNAs, miR-424 and miR-503, it could exert its function acting as reservoir for these microRNAs. However, the effects of miR-503 and miR-424 loss and gain of function experiments on fibrosis measures were minimal compared to the effects after H19X silencing indicating that H19X employs another main molecular mechanism. Indeed, in situ hybridization showed that TGFβ-induced expression of H19X is localized to a defined spot in the nucleus indicating that H19X could influence gene expression by directly interacting with the chromatin. Conclusions H19X is a novel lncRNA which is a key factor in mediating the pro-fibrotic effects of TGFβ in systemic sclerosis and other fibrotic diseases. Disclosure of Interest E. Pachera: None declared, S. Assassi: None declared, G. Salazar: None declared, M. Frank-Bertoncelj: None declared, R. Dobrota: None declared, M. Brock: None declared, F. Kurreeman: None declared, J. de Vries-Bouwstra: None declared, T. Messemaker: None declared, C. Feghali-Bostwick: None declared, J. Distler: None declared, G. Kania: None declared, O. Distler Grant/research support from: from Bayer, Sanofi, Ergonex, Boehringer Ingelheim, Actelion, Pfizer, Consultant for: from 4 D Science, Actelion, Active Biotec, Bayer, BiogenIdec, BMS, Boehringer Ingelheim, EpiPharm, Ergonex, espeRare foundation, Genentech/Roche, GSK, Inventiva, Lilly, medac, MedImmune, Pharmacyclics, Pfizer, Serodapharm, Sinoxa,