Project description:In 15 patients with rheumatoid arthritis the baseline expression (t=0) of type I IFN-regulated genes was determined in peripheral blood cells (PAXgene) using cDNA-microarrays and compared to levels one month after (t=1) anti-TNF treatment (infliximab). Therefore, we used 43K cDNA microarrays from the Stanford Functional Genomics Facility (http://microarray.org/sfgf/) printed on aminosilane-coated slides containing ~20.000 unique genes. Only one batch of arrays was used for all experiments. First DNA spots were UV-cross linked to the slide using 150-300 mJoules. Sample preparation and microarray hybridization were performed as described previously (1,2). Data storage and filtering was performed using the Stanford Microarray Database (http://genome-www5.stanford.edu//) as described previously. 1. van der Pouw Kraan TC, Wijbrandts CA, van Baarsen LG, Voskuyl AE, Rustenburg F, Baggen JM et al.: Rheumatoid arthritis subtypes identified by genomic profiling of peripheral blood cells: assignment of a type I interferon signature in a subpopulation of patients. Ann Rheum Dis 2007, 66: 1008-1014. 2. van der Pouw Kraan TC, van Baarsen LG, Rustenburg F, Baltus B, Fero M, Verweij CL: Gene expression profiling in rheumatology. Methods Mol Med 2007, 136: 305-327. Abbreviations: - Bnrs are the different patients analyzed - PAX or Paxgene indicates the total RNA was isolated from peripheral blood obtained in PAXgene tubes - CRS: common reference sample: We made a common reference that consisted of a mixture of mRNAs isolated from 11 different cell lines (Perou CM, Jeffrey SS, van de RM et al. Distinctive gene expression patterns in human mammary epithelial cells and breast cancers. Proc Natl Acad Sci U S A. 1999; 96:9212-9217) supplemented with RNA from rheumatoid synovial tissue, fibroblasts, and activated peripheral blood mononuclear cells (PBMCs). - PMT: photomultiplier tube: the sensitivity/intensity of the scanner can be adjusted to prevent spot saturation. Compound Based Treatment: anti-TNF (infliximab)

Project description:In 15 patients with rheumatoid arthritis the baseline expression (t=0) of type I IFN-regulated genes was determined in peripheral blood cells (PAXgene) using cDNA-microarrays and compared to levels one month after (t=1) anti-TNF treatment (infliximab). Therefore, we used 43K cDNA microarrays from the Stanford Functional Genomics Facility (http://microarray.org/sfgf/) printed on aminosilane-coated slides containing ~20.000 unique genes. Only one batch of arrays was used for all experiments. First DNA spots were UV-cross linked to the slide using 150-300 mJoules. Sample preparation and microarray hybridization were performed as described previously (1,2). Data storage and filtering was performed using the Stanford Microarray Database (http://genome-www5.stanford.edu//) as described previously. 1. van der Pouw Kraan TC, Wijbrandts CA, van Baarsen LG, Voskuyl AE, Rustenburg F, Baggen JM et al.: Rheumatoid arthritis subtypes identified by genomic profiling of peripheral blood cells: assignment of a type I interferon signature in a subpopulation of patients. Ann Rheum Dis 2007, 66: 1008-1014. 2. van der Pouw Kraan TC, van Baarsen LG, Rustenburg F, Baltus B, Fero M, Verweij CL: Gene expression profiling in rheumatology. Methods Mol Med 2007, 136: 305-327. Abbreviations: - Bnrs are the different patients analyzed - PAX or Paxgene indicates the total RNA was isolated from peripheral blood obtained in PAXgene tubes - CRS: common reference sample: We made a common reference that consisted of a mixture of mRNAs isolated from 11 different cell lines (Perou CM, Jeffrey SS, van de RM et al. Distinctive gene expression patterns in human mammary epithelial cells and breast cancers. Proc Natl Acad Sci U S A. 1999; 96:9212-9217) supplemented with RNA from rheumatoid synovial tissue, fibroblasts, and activated peripheral blood mononuclear cells (PBMCs). - PMT: photomultiplier tube: the sensitivity/intensity of the scanner can be adjusted to prevent spot saturation. Compound Based Treatment: anti-TNF (infliximab) reference_design

Project description:BackgroundSynovial fibroblasts (SFs) are specialized cells of the synovium that provide nutrients and lubricants for the proper function of diarthrodial joints. Recent evidence appreciates the contribution of SF heterogeneity in arthritic pathologies. However, the normal SF profiles and the molecular networks that govern the transition from homeostatic to arthritic SF heterogeneity remain poorly defined.MethodsWe applied a combined analysis of single-cell (sc) transcriptomes and epigenomes (scRNA-seq and scATAC-seq) to SFs derived from naïve and hTNFtg mice (mice that overexpress human TNF, a murine model for rheumatoid arthritis), by employing the Seurat and ArchR packages. To identify the cellular differentiation lineages, we conducted velocity and trajectory analysis by combining state-of-the-art algorithms including scVelo, Slingshot, and PAGA. We integrated the transcriptomic and epigenomic data to infer gene regulatory networks using ArchR and custom-implemented algorithms. We performed a canonical correlation analysis-based integration of murine data with publicly available datasets from SFs of rheumatoid arthritis patients and sought to identify conserved gene regulatory networks by utilizing the SCENIC algorithm in the human arthritic scRNA-seq atlas.ResultsBy comparing SFs from healthy and hTNFtg mice, we revealed seven homeostatic and two disease-specific subsets of SFs. In healthy synovium, SFs function towards chondro- and osteogenesis, tissue repair, and immune surveillance. The development of arthritis leads to shrinkage of homeostatic SFs and favors the emergence of SF profiles marked by Dkk3 and Lrrc15 expression, functioning towards enhanced inflammatory responses and matrix catabolic processes. Lineage inference analysis indicated that specific Thy1+ SFs at the root of trajectories lead to the intermediate Thy1+/Dkk3+/Lrrc15+ SF states and culminate in a destructive and inflammatory Thy1- SF identity. We further uncovered epigenetically primed gene programs driving the expansion of these arthritic SFs, regulated by NFkB and new candidates, such as Runx1. Cross-species analysis of human/mouse arthritic SF data determined conserved regulatory and transcriptional networks.ConclusionsWe revealed a dynamic SF landscape from health to arthritis providing a functional genomic blueprint to understand the joint pathophysiology and highlight the fibroblast-oriented therapeutic targets for combating chronic inflammatory and destructive arthritic disease.

Project description:BackgroundThe adaptive immune response in rheumatoid arthritis (RA) is influenced by an interaction between host genetics and environment, particularly the host microbiome. Association of the gut microbiota with various diseases has been reported, though the specific components of the microbiota that affect the host response leading to disease remain unknown. However, there is limited information on the role of gut microbiota in RA. In this study we aimed to define a microbial and metabolite profile that could predict disease status. In addition, we aimed to generate a humanized model of arthritis to confirm the RA-associated microbe.MethodsTo identify an RA biomarker profile, the 16S ribosomal DNA of fecal samples from RA patients, first-degree relatives (to rule out environment/background as confounding factors), and random healthy non-RA controls were sequenced. Analysis of metabolites and their association with specific taxa was performed to investigate a potential mechanistic link. The role of an RA-associated microbe was confirmed using a human epithelial cell line and a humanized mouse model of arthritis.ResultsPatients with RA exhibited decreased gut microbial diversity compared with controls, which correlated with disease duration and autoantibody levels. A taxon-level analysis suggested an expansion of rare taxa, Actinobacteria, with a decrease in abundant taxa in patients with RA compared with controls. Prediction models based on the random forests algorithm suggested that three genera, Collinsella, Eggerthella, and Faecalibacterium, segregated with RA. The abundance of Collinsella correlated strongly with high levels of alpha-aminoadipic acid and asparagine as well as production of the proinflammatory cytokine IL-17A. A role for Collinsella in altering gut permeability and disease severity was confirmed in experimental arthritis.ConclusionsThese observations suggest dysbiosis in RA patients resulting from the abundance of certain rare bacterial lineages. A correlation between the intestinal microbiota and metabolic signatures could determine a predictive profile for disease causation and progression.

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Anti-TNF drives regulatory T cell expansion by paradoxically promoting membrane TNF-TNF-RII binding in rheumatoid arthritis.

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Anti-TNF drives regulatory T cell expansion by paradoxically promoting membrane TNF-TNF-RII binding in rheumatoid arthritis.

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