Although there have been major advances in the treatment of rheumatoid arthritis with the advent of biological agents, the mechanisms that drive cytokine production and sustain disease chronicity remain unknown. Tenascin-C (encoded by Tnc) is an extracellular matrix glycoprotein specifically expressed at areas of inflammation and tissue damage in inflamed rheumatoid joints. Here we show that mice that do not express tenascin-C show rapid resolution of acute joint inflammation and are protected from erosive arthritis. Intra-articular injection of tenascin-C promotes joint inflammation in vivo in mice, and addition of exogenous tenascin-C induces cytokine synthesis in explant cultures from inflamed synovia of individuals with rheumatoid arthritis. Moreover, in human macrophages and fibroblasts from synovia of individuals with rheumatoid arthritis, tenascin-C induces synthesis of proinflammatory cytokines via activation of Toll-like receptor 4 (TLR4). Thus, we have identified tenascin-C as a novel endogenous activator of TLR4-mediated immunity that mediates persistent synovial inflammation and tissue destruction in arthritic joint disease.

Toll-like receptors (TLRs) and their downstream signaling pathways have been comprehensively characterized in innate immunity. In addition to this function, these receptors have also been suggested to be involved in the pathogenesis of many autoimmune diseases, including rheumatoid arthritis (RA). Murine in vivo models and human in vitro tissue models of RA have provided a wealth of information on the potential activity of TLRs and components of the downstream signaling pathways. Whilst most early work investigated the cell surface TLRs, more recently the focus has moved to the endosomal TLRs 3, 7, 8, and 9. These receptors recognize self and foreign double-stranded RNA and single-stranded RNA and DNA. The development of therapeutics to inhibit the endosomal TLRs or components of their signaling cascades may represent a way to target inflammation upstream of cytokine production. This may allow for greater specificity than existing therapies including cytokine blockade. Here, we review the current information suggesting a role for the endosomal TLRs in RA pathogenesis and the efforts to target these receptors therapeutically.

The mechanism by which oxidative stress induces inflammation and vice versa is unclear but is of great importance, being apparently linked to many chronic inflammatory diseases. We show here that inflammatory stimuli induce release of oxidized peroxiredoxin-2 (PRDX2), a ubiquitous redox-active intracellular enzyme. Once released, the extracellular PRDX2 acts as a redoxdependent inflammatory mediator, triggering macrophages to produce and release TNF-α. The oxidative coupling of glutathione (GSH) to PRDX2 cysteine residues (i.e., protein glutathionylation) occurs before or during PRDX2 release, a process central to the regulation of immunity. We identified PRDX2 among the glutathionylated proteins released in vitro by LPS-stimulated macrophages using mass spectrometry proteomic methods. Consistent with being part of an inflammatory cascade, we find that PRDX2 then induces TNF-α release. Unlike classical inflammatory cytokines, PRDX2 release does not reflect LPS-mediated induction of mRNA or protein synthesis; instead, PRDX2 is constitutively present in macrophages, mainly in the reduced form, and is released in the oxidized form on LPS stimulation. Release of PRDX2 is also observed in human embryonic kidney cells treated with TNF-α. Importantly, the PRDX2 substrate thioredoxin (TRX) is also released along with PRDX2, enabling an oxidative cascade that can alter the -SH status of surface proteins and thereby facilitate activation via cytokine and Toll-like receptors. Thus, our findings suggest a model in which the release of PRDX2 and TRX from macrophages can modify the redox status of cell surface receptors and enable induction of inflammatory responses. This pathway warrants further exploration as a potential novel therapeutic target for chronic inflammatory diseases.cysteine oxidation | thiol oxidation | redox proteomics

IntroductionThe development of novel therapies for sepsis depends on the understanding of the basic mechanisms of the disease. 1 The principal active agent involved in the pathogenesis of sepsis is bacterial lipopolysaccharide (LPS), an essential component of the surface of gram-negative bacteria. LPS exerts its toxic effects by potently activating macrophages and endothelial cells, and inducing the expression of inflammatory cytokines such as tumor necrosis factor ␣ (TNF␣) and interleukin 6 (IL-6). [2][3][4][5] Thus, elucidating how LPS signals through cell-surface receptors to induce inflammatory gene expression in humans is of major importance.Central to the recognition of LPS and also many other microbial products by the host is a family of transmembrane proteins that have leucine-rich repeats in their extracellular domains known as the toll-like receptors (TLRs). 6 LPS interacts with a heterologous receptor that contains TLR4 7,8 as well as CD14 9,10 and MD2. [11][12][13] As CD14 is a glycosyl phosphatidylinositol-anchored protein and MD2 is on the cell surface, transduction of the LPS signal across the membrane is mediated by TLR4. TLR4, as all TLR family members, contains a cytoplasmic domain that is homologous to a cytoplasmic domain found in the IL-1 receptor known as the Toll/IL-1 receptor (IL-1R) homology (TIR) domain that is essential for downstream signaling. [14][15][16] The presence of the TIR domain in both TLR and IL-1 receptor family members suggested that these receptors use an identical framework of signaling molecules to exert their downstream effects. This was supported by subsequent studies in mouse and human cell lines. Thus, IL-1R and TLR4 were shown to engage the TIR-containing cytosolic adaptor molecule myeloid differentiation protein 88 (MyD88) through homotypic interactions, [17][18][19] with subsequent recruitment of IL-1R-associated kinase (IRAK) and IRAK2, IRAK4, and TRAF6. 17,18,20,21 TRAF6 is thought to subsequently activate nuclear factor (NF)-B either through the IB kinase (IKK) complex and the kinases TAB-1 and TAK-1, 22 or through evolutionarily conserved signaling intermediate in Toll pathways (ECSIT) and mitogen-activated protein kinase/ERK kinase kinase 1 (MEKK-1). 23 The recent derivation of MyD88 Ϫ/Ϫ mice, however, challenged a universal role for MyD88 in LPS signaling. Although there was the expected complete ablation of IL-1 signaling, LPS still activated NF-B although the ability to induce TNF␣ from macrophages was lost. 24 In addition, LPS-induced NF-B activation and up-regulation of costimulatory molecules in bone marrowderived dendritic cells from these mice was not compromised. 25 To account for a MyD88-independent pathway of NF-B activation, a novel MyD88 homologue termed MyD88 adaptor-like (Mal) 26 or TIR domain-containing adaptor protein (TIRAP) 27 was described. This was shown to act as an adaptor protein specifically involved in TLR4 but not other TLRs or IL-1R-induced NF-B activation. 26,27 As Mal/TIRAP does not contain the death domain (DD) found in MyD88...

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TLR3 recognizes double-stranded RNA, a product associated with viral infections. Many details of TLR3-induced mechanisms have emerged from gene-targeted mice or inhibition studies in transformed cell lines. However, the pathways activated in human immune cells or cells from disease tissue are less well understood. We have investigated TLR3-induced mechanisms of human primary cells of the innate immune system, including dendritic cells (DCs), macrophages (MØs), endothelial cells (ECs), and synovial fibroblasts isolated from rheumatoid arthritis joint tissue (RA-SFs). Here, we report that while these cells all express TLR3, they differ substantially in their response to TLR3 stimulation. The key antiviral response chemokine IP-10 was produced by all cell types, while DCs and MØs failed to produce the proinflammatory cytokines TNF␣ and IL-6. Unexpectedly, TNF␣ was found secreted by TLR3-stimulated RA-SF. Furthermore, TLR3 stimulation did not activate NFB, MAPKs, or IRF-3 in DCs and MØs, but was able to do so in ECs and RA-SF. These findings were specific for human cells, thereby revealing a complexity not previously expected. This is the first report of such cell type-and species-specific response for any TLR stimulation and helps to explain important difficulties in correlating murine models of inflammatory diseases and human inflammation. IntroductionThe discovery of Toll-like receptors (TLRs) uncovered a key mechanism used by the immune system to detect infections and tissue damage. 1 There are 10 known human TLRs that recognize "molecular patterns" produced as a result of pathogenic infections. TLR3 is a member of this receptor family that is activated by double-stranded RNA (dsRNA), 2 an intermediate formed by most viruses during their replication. The viral dsRNA functions as a danger signal released in the extracellular environment from dying virally infected cells, alerting inflammatory cells and contributing to systemic disease. 3,4 In addition to microbial products, there is growing evidence that TLRs also recognize endogenous ligands found at sites of tissue destruction and cell death, 5 and TLR3 has been shown to recognize mRNA released from dying cells. 6 Although there are concerns that some of the data on endogenous ligands could be a result of contamination with microbial products, the detection of endogenous ligands would be useful in alerting the host of the presence of tissue injury induced by infection or other means. However, there is a potential drawback in that the same endogenous ligands are also found at sites of chronic inflammation and could further drive the inflammatory response in a TLRdependent manner. There is increasing evidence that TLRs could be associated with chronic inflammatory diseases such as rheumatoid arthritis (RA). 5,7 In this context, TLR3 expression is increased in the RA synovium, 8,9 and the presence of TLR3 ligands might affect several aspects of the disease by acting on cells within the joint. In support of this, dsRNA can induce joint inflammation and cause a...

Objective. Selective serotonin reuptake inhibitors (SSRIs), in addition to their antidepressant effects, have been reported to have antiinflammatory effects. The aim of this study was to assess the antiarthritic potential of 2 SSRIs, fluoxetine and citalopram, in murine collagen-induced arthritis (CIA) and in a human ex vivo disease model of rheumatoid arthritis (RA).Methods. Following therapeutic administration of SSRIs, paw swelling was assessed and clinical scores were determined daily in DBA/1 mice with CIA. Joint architecture was examined histologically at the end of the treatment period. Cultures of human RA synovial membranes were treated with SSRIs, and cytokine production was measured. Toll-like receptor (TLR) function was examined in murine and human macrophages, human B cells, and human fibroblast-like synovial cells treated with SSRIs.

The advent of anti-TNF biologicals has been a seminal advance in the treatment of rheumatoid arthritis (RA) and has confirmed the important role of TNF in disease pathogenesis. However, it is unknown what sustains the chronic production of TNF. In this study, we have investigated the anti-inflammatory properties of mianserin, a serotonin receptor antagonist. We discovered mianserin was able to inhibit the endosomal TLRs 3, 7, 8, and 9 in primary human cells and inhibited the spontaneous release of TNF and IL-6 from RA synovial membrane cultures. This suggested a role for these TLRs in production of TNF and IL-6 from RA which was supported by data from chloroquine, an inhibitor of endosomal acidification (a prerequisite for TLRs 3, 7, 8, and 9 activation) which also inhibited production of these cytokines from RA synovial cultures. Only stimulation of TLR 3 or 8 induced TNF from these cultures, indicating that TLR7 and TLR9 were of less consequence in this model. The key observation that indicated the importance of TLR8 was the inhibition of spontaneous TNF production by imiquimod, which we discovered to be an inhibitor of TLR8. Together, these data suggest that TLR8 may play a role in driving TNF production in RA. Because this receptor can be inhibited by small m.w. molecules, it may prove to be an important therapeutic target.