Julia J. Inglis

Tenascin-C is an endogenous activator of Toll-like receptor 4 that is essential for maintaining inflammation in arthritic joint disease.

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.

Blockade of tumor necrosis factor in collagen-induced arthritis reveals a novel immunoregulatory pathway for Th1 and Th17 cells.

IL-17 is implicated in the pathogenesis of rheumatoid arthritis (RA) and has previously been shown to be induced by tumor necrosis factor (TNF) in vitro. The aim of this study was to assess the impact of TNF inhibition on IL-17 production in collagen-induced arthritis, a model of RA. TNF blockade using TNFR-Fc fusion protein or anti-TNF monoclonal antibody reduced arthritis severity but, unexpectedly, expanded populations of Th1 and Th17 cells, which were shown by adoptive transfer to be pathogenic. Th1 and Th17 cell populations were also expanded in collagen-immunized TNFR p55−/− but not p75−/− mice. The expression of IL-12/IL-23 p40 was up-regulated in lymph nodes (LN) from p55−/− mice, and the expansion of Th1/Th17 cells was abrogated by blockade of p40. Treatment of macrophages with rTNF also inhibited p40 production in vitro. These findings indicate that at least one of the ways in which TNF regulates Th1/Th17 responses in arthritis is by down-regulating the expression of p40. Finally, although TNF blockade increased numbers of Th1 and Th17 cells in LN, it inhibited their accumulation in the joint, thereby providing an explanation for the paradox that anti-TNF therapy ameliorates arthritis despite increasing numbers of pathogenic T cells.

Fibroblast growth factor 2 is an intrinsic chondroprotective agent that suppresses ADAMTS‐5 and delays cartilage degradation in murine osteoarthritis

OBJECTIVE We have previously identified in articular cartilage an abundant pool of the heparin-binding growth factor, fibroblast growth factor 2 (FGF-2), which is bound to the pericellular matrix heparan sulfate proteoglycan, perlecan. This pool of FGF-2 activates chondrocytes upon tissue loading and is released following mechanical injury. In vitro, FGF-2 suppresses interleukin-1-driven aggrecanase activity in human cartilage explants, suggesting a chondroprotective role in vivo. We undertook this study to investigate the in vivo role of FGF-2 in murine cartilage. METHODS Basal characteristics of the articular cartilage of Fgf2(-/-) and Fgf2(+/+) mice were determined by histomorphometry, nanoindentation, and quantitative reverse transcriptase-polymerase chain reaction. The articular cartilage was graded histologically in aged mice as well as in mice in which osteoarthritis (OA) had been induced by surgical destabilization of the medial meniscus. RNA was extracted from the joints of Fgf2(-/-) and Fgf2(+/+) mice following surgery and quantitatively assessed for key regulatory molecules. The effect of subcutaneous administration of recombinant FGF-2 on OA progression was assessed in Fgf2(-/-) mice. RESULTS Fgf2(-/-) mice were morphologically indistinguishable from wild-type (WT) animals up to age 12 weeks; the cartilage thickness and proteoglycan staining were equivalent, as was the mechanical integrity of the matrix. However, Fgf2(-/-) mice exhibited accelerated spontaneous and surgically induced OA. Surgically induced OA in Fgf2(-/-) mice was suppressed to levels in WT mice by subcutaneous administration of recombinant FGF-2. Increased disease in Fgf2(-/-) mice was associated with increased expression of messenger RNA of Adamts5, the key murine aggrecanase. CONCLUSION These data identify FGF-2 as a novel endogenous chondroprotective agent in articular cartilage.

Protocol for the induction of arthritis in C57BL/6 mice

Collagen-induced arthritis is a well-validated, but strain-dependent mouse model of rheumatoid arthritis, with H-2q and H-2r strains showing the greatest degree of susceptibility. This protocol describes the induction of arthritis in the C57BL/6 strain (H-2b), which forms the genetic background of the majority of genetically modified strains. This protocol involves purification of type II collagen from chicken sternums, immunization of mice, clinical assessment of arthritis and analysis of T- and B-cell responses to type II collagen. Key aspects of the protocol are the need to use chicken collagen for immunization and the importance of avoiding aggressive behavior in males. The incidence of arthritis varies from 50 to 80% and is milder than the classical collagen-induced arthritis model. This procedure takes ∼3 months to complete.

Apremilast, a novel PDE4 inhibitor, inhibits spontaneous production of tumour necrosis factor-alpha from human rheumatoid synovial cells and ameliorates experimental arthritis.

IntroductionType 4 phosphodiesterases (PDE4) play an important role in immune cells through the hydrolysis of the second messenger, cAMP. Inhibition of PDE4 has previously been shown to suppress immune and inflammatory responses, demonstrating PDE4 to be a valid therapeutic target for immune-mediated pathologies. We assessed the anti-inflammatory effects of a novel PDE4 inhibitor, apremilast, in human synovial cells from rheumatoid arthritis (RA) patients, as well as two murine models of arthritis.MethodsCells liberated from tissue excised from arthritic joints of RA patients were cultured in the presence of increasing concentrations of apremilast for 48 hours and spontaneous tumour necrosis factor-alpha (TNFα) production was analysed in culture supernatants by ELISA. In addition, arthritis was induced in BALB/c and DBA/1 mice by passive transfer of anti-type II collagen mAb and immunisation with type II collagen, respectively. Mice with established arthritis received 5 or 25 mg/kg apremilast and disease severity was monitored relative to mice receiving vehicle alone. At the end of the study, paws were removed and processed for histopathological assessment. Behavioural effects of apremilast, relative to rolipram, were assessed in naïve DBA/1 mice using an automated activity monitor (LABORAS).ResultsApremilast dose dependently inhibited spontaneous release of TNFα from human rheumatoid synovial membrane cultures. Furthermore, apremilast significantly reduced clinical score in both murine models of arthritis over a ten day treatment period and maintained a healthy joint architecture in a dose-dependent manner. Importantly, unlike rolipram, apremilast demonstrated no adverse behavioural effects in naïve mice.ConclusionsApremilast is an orally available PDE4 inhibitor that reduces TNFα production from human synovial cells and significantly suppresses experimental arthritis. Apremilast appears to be a potential new agent for the treatment of rheumatoid arthritis.

Collagen-induced arthritis in C57BL/6 mice is associated with a robust and sustained T-cell response to type II collagen

Many genetically modified mouse strains are now available on a C57BL/6 (H-2b) background, a strain that is relatively resistant to collagen-induced arthritis. To facilitate the molecular understanding of autoimmune arthritis, we characterised the induction of arthritis in C57BL/6 mice and then validated the disease as a relevant pre-clinical model for rheumatoid arthritis.C57BL/6 mice were immunised with type II collagen using different protocols, and arthritis incidence, severity, and response to commonly used anti-arthritic drugs were assessed and compared with DBA/1 mice. We confirmed that C57BL/6 mice are susceptible to arthritis induced by immunisation with chicken type II collagen and develop strong and sustained T-cell responses to type II collagen. Arthritis was milder in C57BL/6 mice than DBA/1 mice and more closely resembled rheumatoid arthritis in its response to therapeutic intervention. Our findings show that C57BL/6 mice are susceptible to collagen-induced arthritis, providing a valuable model for assessing the role of specific genes involved in the induction and/or maintenance of arthritis and for evaluating the efficacy of novel drugs, particularly those targeted at T cells.

Treatment of murine osteoarthritis with TrkAd5 reveals a pivotal role for nerve growth factor in non-inflammatory joint pain.

&NA; The origin of pain in osteoarthritis is poorly understood, but it is generally thought to arise from inflammation within the innervated structures of the joint, such as the synovium, capsule and bone. We investigated the role of nerve growth factor (NGF) in pain development in murine OA, and the analgesic efficacy of the soluble NGF receptor, TrkAD5. OA was induced in mice by destabilisation of the medial meniscus and pain was assessed by measuring hind‐limb weight distribution. RNA was extracted from joints, and NGF and TNF expressions were quantified. The effect of tumour necrosis factor (TNF) and neutrophil blockade on NGF expression and pain were also assessed. NGF was induced in the joints during both post‐operative (day 3) and OA (16 weeks) pain, but not in the non‐painful stage of disease (8 weeks post‐surgery). TrkAd5 was highly effective at suppressing pain in both phases. Induction of NGF in the post‐operative phase of pain was TNF‐dependent as anti‐TNF reduced NGF expression in the joint and abrogated pain. However, TNF was not regulated in the late OA joints, and pain was not affected by anti‐TNF therapy. Fucoidan, by suppressing cellular infiltration into the joint, was able to suppress post‐operative, but not late OA pain. These results indicate that NGF is an important mediator of OA pain and that TrkAd5 represents a potent novel analgesic in this condition. They also suggest that, unlike post‐operative pain, induction of pain in OA may not necessarily be driven by classical inflammatory processes.

Regulation of pain sensitivity in experimental osteoarthritis by the endogenous peripheral opioid system

OBJECTIVE OA is the most common joint disease, affecting 10-15% of people over 60 years of age. However, up to 40% of individuals with radiologic damage are asymptomatic. The purpose of this study was to assess the role of the endogenous opioid system in delaying the onset of pain in a murine model of osteoarthritis (OA). METHODS Osteoarthritis was induced by transection of the medial meniscotibial ligament. Pain was assessed by monitoring weight distribution and activity. At various times postsurgery, the opioid receptor antagonists naloxone or peripherally restricted naloxone methiodide were administered, and pain was assessed. Levels of the micro-opioid receptor were assessed in the nerves innervating the joint by real-time reverse transcription-polymerase chain reaction analysis. RESULTS As in human disease, significant joint damage occurred in mice before the onset of pain. To assess whether delayed pain was partly the result of increased endogenous opioid function, naloxone or naloxone methiodide was administered. Both opioid receptor antagonists led to pain onset 4 weeks earlier than in vehicle-treated mice, indicating a role of the peripheral opioid system in masking OA pain. The expression of the micro-opioid receptor in the peripheral nerves supplying the joint was transiently increased in naloxone-responsive mice. CONCLUSION These findings indicate that a temporal induction of micro-opioid receptors in the early stages of OA delays the onset of pain. This is of clinical relevance and may contribute to the assessment of patients presenting with pain late in the disease. Furthermore, it may point to a mechanism by which the body blocks pain perception in moderate states of tissue damage, allowing an increased chance of survival.

Indoleamine 2,3 dioxygenase-mediated tryptophan catabolism regulates accumulation of Th1/Th17 cells in the joint in collagen-induced arthritis

OBJECTIVE Indoleamine 2,3 dioxygenase (IDO) is a catabolic enzyme that initiates the kynurenine pathway of tryptophan degradation and has immunomodulatory properties. The aim of this study was to investigate the regulation of collagen-induced arthritis by tryptophan catabolism mediated by IDO. METHODS Arthritis was induced by immunization with type II collagen. After induction of arthritis, the expression of IDO was analyzed by quantitative reverse transcription-polymerase chain reaction. The effect of IDO deficiency on collagen-induced arthritis was assessed in vivo by administration of 1-methyltryptophan and clinical and histologic evaluation of IDO-deficient mice. The requirement for IDO activation was bypassed by administration of L-kynurenine. RESULTS IDO was induced in lymph node dendritic cells after collagen immunization. Systemic inhibition of tryptophan catabolism during active arthritis increased disease severity. Conversely, bypassing the requirement for tryptophan degradation by the administration of L-kynurenine resulted in amelioration of arthritis. Furthermore, IDO-deficient mice showed a higher incidence of arthritis and exacerbated disease severity compared with IDO-competent mice. Such increased disease activity in IDO-deficient mice correlated early with increased production of the proinflammatory cytokines interferon-gamma and interleukin-17 by lymph node T cells and later with increased infiltration of Th1 and Th17 cells in the inflamed joints. CONCLUSION Our data indicate that the induction of IDO controls the accumulation of Th1 and Th17 pathogenic T cells at the site of inflammation during collagen-induced arthritis. Therefore, manipulation of the kynurenine pathway of tryptophan degradation provides the potential for therapeutic intervention in rheumatoid arthritis.

Anti-CD3 Therapy Expands the Numbers of CD4+ and CD8+ Treg Cells and Induces Sustained Amelioration of Collagen-Induced Arthritis

OBJECTIVE To assess the therapeutic potential of anti-CD3 monoclonal antibodies (mAb) for rheumatoid arthritis, using collagen-induced arthritis as an animal model. METHODS Arthritis was induced in DBA/1 mice by immunization with type II collagen. After disease onset, a single injection of anti-CD3 mAb (20 microg/mouse) was administered, and arthritis severity was monitored over a 10-day period. RESULTS Anti-CD3 mAb treatment resulted in a sustained reduction in disease activity, which was associated with an increase in the proportion of naturally occurring CD4+CD25+FoxP3+ regulatory T (Treg) cells and the generation of a population of CD8+CD25+FoxP3+ Treg cells. Anti-CD3 mAb treatment did not alter the capacity of CD4+ Treg cells to suppress effector T cell proliferation and interferon-gamma (IFNgamma) production in vitro. However, CD4+ Treg cells from both anti-CD3 mAb-treated and control mice were unable to suppress interleukin-17 (IL-17) production. In contrast, CD8+ Treg cells induced by anti-CD3 therapy suppressed IL-17 production as well as CD4+ T cell proliferation and IFNgamma production. CONCLUSION These results show that anti-CD3 mAb treatment has important therapeutic potential for rheumatoid arthritis and has the capacity to generate antiarthritic CD8+ Treg cells and expand the relative numbers of CD4+ Treg cells.