Functional analysis of an arthritogenic synovial fibroblast

Increasing attention has been directed towards identifying non-T-cell mechanisms as potential therapeutic targets in rheumatoid arthritis. Synovial fibroblast (SF) activation, a hallmark of rheumatoid arthritis, results in inappropriate production of chemokines and matrix components, which in turn lead to bone and cartilage destruction. We have demonstrated that SFs have an autonomous pathogenic role in the development of the disease, by showing that they have the capacity to migrate throughout the body and cause pathology specifically to the joints. In order to decipher the pathogenic mechanisms that govern SF activation and pathogenic potential, we used the two most prominent methods of differential gene expression analysis, differential display and DNA microarrays, in a search for deregulated cellular pathways in the arthritogenic SF. Functional clustering of differentially expressed genes, validated by dedicated in vitro functional assays, implicated a number of cellular pathways in SF activation. Among them, diminished adhesion to the extracellullar matrix was shown to correlate with increased proliferation and migration to this matrix. Our findings support an aggressive role for the SF in the development of the disease and reinforce the perspective of a transformed-like character of the SF

MUGEN mouse database; Animal models of human immunological diseases

The MUGEN mouse database (MMdb) (www.mugen-noe.org/database/) is a database of murine models of immune processes and immunological diseases. Its aim is to share and publicize information on mouse strain characteristics and availability from participating institutions. MMdb's basic classification of models is based on three major research application categories: Models of Human Disease, Models of Immune Processes and Transgenic Tools. Data on mutant strains includes detailed information on affected gene(s), mutant allele(s) and genetic background (DNA origin, gene targeted, host and backcross strain background). Each gene/transgene index also includes IDs and direct links to Ensembl, ArrayExpress, EURExpress and NCBI's Entrez Gene database. Phenotypic description is standardized and hierarchically structured, based on MGI's mammalian phenotypic ontology terms. Availability (e.g. live mice, cryopreserved embryos, sperm and ES cells) is clearly indicated, along with handling and genotyping details (in the form of documents or hyperlinks) and all relevant contact information (including EMMA and Jax/IMSR hyperlinks where available). MMdb's design offers a user-friendly query interface and provides instant access to the list of mutant strains and genes. Database access is free of charge and there are no registration requirements for data querying

A20 Prevents Inflammasome-Dependent Arthritis by Inhibiting Macrophage Necroptosis Through Its ZnF7 Ubiquitin-Binding Domain

Deficiency in the deubiquitinating enzyme A20 causes severe inflammation in mice, and impaired A20 function is associated with human inflammatory diseases. A20 has been implicated in negatively regulating NF-κB signalling, cell death and inflammasome activation; however, the mechanisms by which A20 inhibits inflammation in vivo remain poorly understood. Genetic studies in mice revealed that its deubiquitinase activity is not essential for A20 anti-inflammatory function. Here we show that A20 prevents inflammasome-dependent arthritis by inhibiting macrophage necroptosis and that this function depends on its zinc finger 7 (ZnF7). We provide genetic evidence that RIPK1 kinase-dependent, RIPK3-MLKL-mediated necroptosis drives inflammasome activation in A20-deficient macrophages and causes inflammatory arthritis in mice. Single-cell imaging revealed that RIPK3-dependent death caused inflammasome-dependent IL-1β release from lipopolysaccharide-stimulated A20-deficient macrophages. Importantly, mutation of the A20 ZnF7 ubiquitin binding domain caused arthritis in mice, arguing that ZnF7-dependent inhibition of necroptosis is critical for A20 anti-inflammatory function in vivo

Brief report: Group 3 innate lymphoid cells in human enthesis

Objective: Group 3 innate lymphoid cells (ILC3s) play a pivotal role in barrier tissues such as the gut and the skin, two important sites of disease in spondyloarthropathy (SpA). It was investigated whether normal and injured human enthesis, the key target tissue in early SpA, harboured ILC3s in entheseal soft tissue (EST) and adjacent peri-entheseal bone (PEB). Methods: Interspinous ligament and spinous process bone was collected from donors with no systemic inflammatory disease, enzymatically digested and immunophenotyped. The immunological profile of entheseal cells was examined and the transcriptional profile of sorted ILC3s was compared to those isolated from SpA synovial fluid. To assess the ability of entheseal tissue to produce IL-17 and IL-22 entheseal digests were stimulated with IL-23 and IL-1β. Osteoarthritic and ruptured Achilles tissue was examined histologically. Results: Compared to peripheral blood, human EST had a higher proportion of ILCs (p=0.008), EST and PEB both had a higher proportion of NKp44+ ILC3s (p=0.001 and p=0.043). RORγt, STAT3 and IL-23R transcript expression validated the entheseal ILC3 phenotype. Cytokine transcript expression was similar in ILC3s isolated from enthesis and SpA synovial fluid. Normal entheseal digests stimulated with IL-23/IL-1β upregulated IL17A transcript and histological examination of injured/damaged entheses showed RORγt expressing cells. Conclusion: This work shows that human enthesis harbours a resident population of ILC3s, with the potential to participate in spondyloarthropathy pathogenesis

Genetic Dissection of the Cellular Pathways and Signaling Mechanisms in Modeled Tumor Necrosis Factor–induced Crohn's-like Inflammatory Bowel Disease

Recent clinical evidence demonstrated the importance of tumor necrosis factor (TNF) in the development of Crohn's disease. A mouse model for this pathology has previously been established by engineering defects in the translational control of TNF mRNA (TnfΔAREmouse). Here, we show that development of intestinal pathology in this model depends on Th1-like cytokines such as interleukin 12 and interferon γ and requires the function of CD8+ T lymphocytes. Tissue-specific activation of the mutant TNF allele by Cre/loxP-mediated recombination indicated that either myeloid- or T cell–derived TNF can exhibit full pathogenic capacity. Moreover, reciprocal bone marrow transplantation experiments using TNF receptor–deficient mice revealed that TNF signals are equally pathogenic when directed independently to either bone marrow–derived or tissue stroma cell targets. Interestingly, TNF-mediated intestinal pathology was exacerbated in the absence of MAPKAP kinase 2, yet strongly attenuated in a Cot/Tpl2 or JNK2 kinase–deficient genetic background. Our data establish the existence of redundant cellular pathways operating downstream of TNF in inflammatory bowel disease, and demonstrate the therapeutic potential of selective kinase blockade in TNF-mediated intestinal pathology

Cytoskeletal Rearrangements in Synovial Fibroblasts as a Novel Pathophysiological Determinant of Modeled Rheumatoid Arthritis

Rheumatoid arthritis is a chronic inflammatory disease with a high prevalence and substantial socioeconomic burden. Despite intense research efforts, its aetiology and pathogenesis remain poorly understood. To identify novel genes and/or cellular pathways involved in the pathogenesis of the disease, we utilized a well-recognized tumour necrosis factor-driven animal model of this disease and performed high-throughput expression profiling with subtractive cDNA libraries and oligonucleotide microarray hybridizations, coupled with independent statistical analysis. This twin approach was validated by a number of different methods in other animal models of arthritis as well as in human patient samples, thus creating a unique list of disease modifiers of potential therapeutic value. Importantly, and through the integration of genetic linkage analysis and Gene Ontology–assisted functional discovery, we identified the gelsolin-driven synovial fibroblast cytoskeletal rearrangements as a novel pathophysiological determinant of the disease

Two distinct ubiquitin-binding motifs in A20 mediate its anti-inflammatory and cell-protective activities

Protein ubiquitination regulates protein stability and modulates the composition of signaling complexes. A20 is a negative regulator of inflammatory signaling, but the molecular mechanisms involved are ill understood. Here, we generated Tnfaip3 gene-targeted A20 mutant mice bearing inactivating mutations in the zinc finger 7 (ZnF7) and ZnF4 ubiquitin-binding domains, revealing that binding to polyubiquitin is essential for A20 to suppress inflammatory disease. We demonstrate that a functional ZnF7 domain was required for recruiting A20 to the tumor necrosis factor receptor 1 (TNFR1) signaling complex and to suppress inflammatory signaling and cell death. The combined inactivation of ZnF4 and ZnF7 phenocopied the postnatal lethality and severe multiorgan inflammation of A20-deficient mice. Conditional tissue-specific expression of mutant A20 further revealed the key role of ubiquitin-binding in myeloid and intestinal epithelial cells. Collectively, these results demonstrate that the anti-inflammatory and cytoprotective functions of A20 are largely dependent on its ubiquitin-binding properties. van Loo and colleagues provide insights into the action of the anti-inflammatory protein A20. The ZnF7 and ZnF4 ubiquitin-binding domains of A20 are both required to suppress inflammatory signaling and cell death; however, these zinc fingers operate via distinct mechanisms

The long non-coding RNA HOTAIR contributes to joint-specific gene expression in rheumatoid arthritis

Although patients with rheumatoid arthritis (RA) typically exhibit symmetrical joint involvement, some patients develop alternative disease patterns in response to treatment, suggesting that different molecular mechanism may underlie disease progression depending on joint location. Here, we identify joint-specific changes in RA synovium and synovial fibroblasts (SF) between knee and hand joints. We show that the long non-coding RNA HOTAIR, which is only expressed in knee SF, regulates more than 50% of this site-specific gene expression in SF. HOTAIR is downregulated after stimulation with pro-inflammatory cytokines and is expressed at lower levels in knee samples from patients with RA, compared with osteoarthritis. Knockdown of HOTAIR in knee SF increases PI-Akt signalling and IL-6 production, but reduces Wnt signalling. Silencing HOTAIR inhibits the migratory function of SF, decreases SF-mediated osteoclastogenesis, and increases the recruitment of B cells by SF. We propose that HOTAIR is an important epigenetic factor in joint-specific gene expression in RA