Cells of the synovium in rheumatoid arthritis. Macrophages

The multitude and abundance of macrophage-derived mediators in rheumatoid arthritis and their paracrine/autocrine effects identify macrophages as local and systemic amplifiers of disease. Although uncovering the etiology of rheumatoid arthritis remains the ultimate means to silence the pathogenetic process, efforts in understanding how activated macrophages influence disease have led to optimization strategies to selectively target macrophages by agents tailored to specific features of macrophage activation. This approach has two advantages: (a) striking the cell population that mediates/amplifies most of the irreversible tissue destruction and (b) sparing other cells that have no (or only marginal) effects on joint damage

Transcriptomic analysis of peripheral blood monocytes and synovial macrophages in inflammatory arthritis

Background: Rheumatoid arthritis (RA) and psoriatic arthritis (PsA) are two distinct forms of chronic auto-immunity; understanding the transcriptomic profiles of key leukocyte subsets implicated in these arthritides could improve the diagnosis and treatment of patients. Current microarray analyses of samples derived from RA and PsA patients have examined the genetic profiles of whole blood or diseased tissue which, although informative, can mask the genetic contributions of individual cell types. Monocytes and macrophages are a cellular subset known to play a major role in PsA and RA through the production of pro-inflammatory chemokines, cytokines and destructive proteinases. Aim: To define the transcriptome in CD14+ cells separated from the blood and synovial fluid of PsA and RA patients, and to then compare and contrast that signature in health and disease. Thereafter to define the relevant activities of selected novel moieties described in the foregoing analysis. Methods & Results: The transcriptomic profiles of healthy, RA and PsA CD14+ blood cells were remarkably similar - few genes could distinguish diseased from healthy CD14+ cells. Comparison of the genetic signature of the RA and PsA synovial fluid CD14+ cells revealed that just over 50% of the differentially expressed genes were shared between the two disease groups. Furthermore, analysing the canonical pathways in the synovial fluid cells compared to the matched peripheral blood of both patient groups surprisingly revealed Liver X receptor (LXR) activation pathway as the most significantly upregulated pathway: this pathway has been previously shown by our group to play a pro-inflammatory role in arthritis. Examination of specific upregulated mRNAs in the synovial fluid CD14+ cells from both disease types revealed two novel genes that had not previously been associated with arthritis, the lysosomal enzyme legumain and the cell surface molecule plexin A1. Legumain was demonstrated to be present in RA and PsA CD14+ cells by RNA and protein analysis and was physiologically active. Incubation of CD14+ cells with patient synovial fluid under hypoxic conditions also potentiated legumain expression. Plexin A1 was confirmed to be expressed at the mRNA level within RA synovium. siRNA knockdown of plexin A1 suggested that it may play a pro-inflammatory role within macrophages since subsequent treatment of these macrophages with LPS resulted in decreased TNFα production. However, investigations into the identity of the specific ligands for plexin A1 in arthritis, known as semaphorins, were inconclusive. I finally generated microarray data to evaluate the transcriptome of macrophages activated via cel contact with activated T cells. Such cells shared only a small percentage of genes with those dysregulated in the RA and PsA synovial fluid derived CD14+ cells suggesting that this model at the time points chosen may not be an appropriate in vitro representation of articular macrophages. An imaging system of this in vitro model was also established to visualise the dynamic nature of the T cell – macrophage interactions and demonstrated that variables such as duration or method of T cell activation could alter the number and duration of interactions between the two cell types. Conclusions: These studies demonstrate that the CD14+ cells isolated from the blood are similar transcriptomically between healthy controls and RA and PsA patients. The synovial fluid CD14+ cells from RA and PsA patients exhibit substantial overlap in terms of their genetic profile. Two novel molecules expressed by diseased patients namely plexin A1 and legumain have been identified and their preliminary characteristics in the context of synovitis have been defined

ADAM‐10 is overexpressed in rheumatoid arthritis synovial tissue and mediates angiogenesis

Objective To examine the expression of ADAM‐10 in rheumatoid arthritis (RA) synovial tissue (ST) and the role it plays in angiogenesis. Methods ADAM‐10 expression was determined using immunohistology, Western blotting, and quantitative polymerase chain reaction. In order to examine the role of ADAM‐10 in angiogenesis, we performed in vitro Matrigel tube formation and chemotaxis assays using human microvascular endothelial cells (HMVECs) transfected with control or ADAM‐10 small interfering RNA (siRNA). To determine whether ADAM‐10 plays a role in angiogenesis in the context of RA, we performed Matrigel assays using a coculture system of HMVECs and RA synovial fibroblasts. Results Endothelial cells and lining cells within RA ST expressed high levels of ADAM‐10 compared with cells within osteoarthritis ST and normal ST. ADAM‐10 expression was significantly elevated at the protein and messenger RNA levels in HMVECs and RA synovial fibroblasts stimulated with proinflammatory mediators compared with unstimulated cells. ADAM‐10 siRNA–treated HMVECs had decreased endothelial cell tube formation and migration compared with control siRNA–treated HMVECs. In addition, ADAM‐10 siRNA–treated HMVECs from the RA synovial fibroblast coculture system had decreased endothelial cell tube formation compared with control siRNA–treated HMVECs. Conclusion These data show that ADAM‐10 is overexpressed in RA and suggest that ADAM‐10 may play a role in RA angiogenesis. ADAM‐10 may be a potential therapeutic target in inflammatory angiogenic diseases such as RA.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/94711/1/37755_ftp.pd

Folate-targeted nanoparticles for rheumatoid arthritis therapy

Rheumatoid arthritis (RA) is the most common inflammatory rheumatic disease, affecting almost 1% of the world population. Although the cause of RA remains unknown, the complex interaction between immune mediators (cytokines and effector cells) is responsible for the joint damage that begins at the synovial membrane. Activated macrophages are critical in the pathogenesis of RA and have been shown to specifically express a receptor for the vitamin folic acid (FA), folate receptor (FR). This particular receptor allows internalization of FA-coupled cargo. In this review we will address the potential of nanoparticles as an effective drug delivery system for therapies that will directly target activated macrophages. Special attention will be given to stealth degree of the nanoparticles as a strategy to avoid clearance by macrophages of the mononuclear phagocytic system (MPS). This review summarizes the application of FA-target nanoparticles as drug delivery systems for RA and proposes prospective future directions.Eugénia Nogueira (SFRH/BD/81269/2011) holds a scholarship from Fundação para a Ciência e a Tecnologia (FCT). This study was funded by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement NMP4-LA-2009-228827 NANOFOL. The authors thank the FCT Strategic Project of UID/BIO/04469/2013 unit, the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) and the Project “BioHealth — Biotechnology and Bioengineering approaches to improve health quality”, Ref. NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON.2 — O Novo Norte), QREN, FEDER. This work was also supported by FCT I.P. through the strategic funding UID/BIA/04050/2013. We thank the Centro Hospitalar do Alto Ave (Guimarães, Portugal) for providing radiographic joint images

The Role of Activated Protein C in Bone, Arthritis, and Fracture Healing

Activated protein C (APC) is a cytoprotective anticoagulant that stimulates cellular proliferation, suppresses inflammation, and enhances wound healing. These properties of APC are primarily modulated through its receptors, endothelial protein C receptor (EPCR) and protease-activated receptors (PAR)1/2, and subsequent activation of downstream proteins including ERK1/2, Akt, and p38. In this study, APC was investigated for its potential application in bone repair and arthritic bone conditions, including rheumatoid arthritis (RA) and osteoarthritis (OA), through its actions on osteoblasts. APC increased viability of MG-63 and MC3T3-E1 cells through a PAR1 dependent pathway and subsequently upregulated pERK, pAkt, and p-p38. APC augmented bone and tissue volume but not osteoclast numbers in a BMP-2 induced murine ectopic bone formation model. APC, however, did not enhance callus formation in a closed murine mid-tibial fracture model owing to several study limitations. In contrast with its effects in osteoblastic cell lines, APC suppressed cell viability through an EPCR/PAR1/PAR2 dependent mechanism in OA and RA bone derived cells. APC also decreased pERK, increased p27, and modulated IL-6 and MMP-2 secretion in arthritic cells. Collectively, these results demonstrate the diverse actions of APC in normal and arthritic bone biology including the novel potential of APC on bone formation

Expression of protease-activated receptors in arthritic synovial tissues

Clinical and experimental evidence suggests that synovial thrombin formation in arthritic joints is prominent and deleterious, leading to exacerbation of rheumatoid arthritis (RA). In this context, cellular effects of thrombin mediated by the protease-activated receptors (PARs) in arthritic joints may be of paramount significance. Four PARs have now been identified. PAR1, PAR3, and PAR4 can all be activated by thrombin whereas PAR2 is activated by trypsin and few other proteases.We first explored PARs expression in RA synovial tissues. Synovial membranes from 11 RA patients were analyzed for PARs expression by RT-PCR and by immunohistology. PAR4 was found in all the biopsies, whereas the expression of PAR1, PAR 2 and PAR3 was more restricted (8/11, 5/11 and 3/11 respectively). In the arthritic synovial membrane of murine antigen-induced arthritis (AIA) we found coexpression of the four different PARs. Next, we explored the functional importance of PAR1 during AIA in vivo using PAR-1 deficient mice. The phenotype of PAR1-deficient mice (n = 22), based on the analysis of arthritis severity (as measured by 99 m tecnetium uptake, histological scoring and intra-articular fibrin measurements) was similar to that of wild-type mice (n = 24). In addition, the in vivo production of antibodies against mBSA was also similar. By contrast, the mBSA-induced in vitro lymph node cell proliferation was significantly decreased in PAR1-deficient mice as compared with controls. Accordingly, mBSA-induced production of interferon-γ by lymph node cells in culture was significantly decreased in PAR1-deficient mice as compared with controls, whereas opposite results were observed for production of IL-10

Th17 cells in human disease

Our understanding of the role of T cells in human disease is undergoing revision as a result of the discovery of T-helper 17 (Th17) cells, a unique CD4 + T-cell subset characterized by production of interleukin-17 (IL-17). IL-17 is a highly inflammatory cytokine with robust effects on stromal cells in many tissues. Recent data in humans and mice suggest that Th17 cells play an important role in the pathogenesis of a diverse group of immune-mediated diseases, including psoriasis, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, and asthma. Initial reports also propose a role for Th17 cells in tumorigenesis and transplant rejection. Important differences, as well as many similarities, are emerging when the biology of Th17 cells in the mouse is compared with corresponding phenomena in humans. As our understanding of human Th17 biology grows, the mechanisms underlying many diseases are becoming more apparent, resulting in a new appreciation for both previously known and more recently discovered cytokines, chemokines, and feedback mechanisms. Given the strong association between excessive Th17 activity and human disease, new therapeutic approaches targeting Th17 cells are highly promising, but the potential safety of such treatments may be limited by the role of these cells in normal host defenses against infection.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72455/1/j.1600-065X.2008.00628.x.pd

Inflammatory properties of inhibitor of DNA binding 1 secreted by synovial fibroblasts in rheumatoid arthritis

Abstract Background Inhibitor of DNA binding 1 (Id1) is a nuclear protein containing a basic helix-loop-helix (bHLH) domain that regulates cell growth by selective binding and prevention of gene transcription. Sources of Id1 production in rheumatoid arthritis synovial tissue (RA ST) and its range of functional effects in RA remain to be clarified. Methods We analyzed Id1 produced from synovial fibroblasts and endothelial cells (ECs) with histology and real-time polymerase chain reaction (RT-PCR). Fibroblast supernatants subjected to differential centrifugation to isolate and purify exosomes were measured for Id1 by enzyme-linked immunosorbent assay (ELISA). Western blotting of Id1-stimulated ECs was performed to determine the kinetics of intracellular protein phosphorylation. EC intracellular signaling pathways induced by Id1 were subsequently targeted with silencing RNA (siRNA) for angiogenesis inhibition. Results By PCR and histologic analysis, we found that the primary source of Id1 in STs is from activated fibroblasts that correlate with inflammatory scores in human RA ST and in joints from K/BxN serum-induced mice. Normal (NL) and RA synovial fibroblasts increase Id1 production with stimulation by transforming growth factor beta (TGF-β). Most of the Id1 released by RA synovial fibroblasts is contained within exosomes. Endothelial progenitor cells (EPCs) and human dermal microvascular ECs (HMVECs) activate the Jnk signaling pathway in response to Id1, and Jnk siRNA reverses Id1-induced HMVEC vessel formation in Matrigel plugs in vivo. Conclusions Id1 is a pleotropic molecule affecting angiogenesis, vasculogenesis, and fibrosis. Our data shows that Id1 is not only an important nuclear protein, but also can be released from fibroblasts via exosomes. The ability of extracellular Id1 to activate signaling pathways expands the role of Id1 in the orchestration of tissue inflammation.http://deepblue.lib.umich.edu/bitstream/2027.42/134552/1/13075_2016_Article_984.pd