TNFR2 signalling in inflammatory diseases.

TNF signals via two receptors, TNFR1 and TNFR2, which play contrasting roles in immunity. Most of the pro-inflammatory effects of TNF are mediated by TNFR1, whereas TNFR2 is mainly involved in immune homeostasis and tissue healing, but also contributes to tumour progression. However, all currently available anti-TNF biologics inhibit signalling via both receptors and there is increasing interest in the development of selective inhibitors; TNFR1 inhibitors for autoimmune disease and TNFR2 inhibitors for cancer. It is hypothesised that selective inhibition of TNFR1 in autoimmune disease would alleviate inflammation and promote homeostasis by allowing TNFR2 signalling to proceed unimpeded. Validation of this concept would pave the way for the development and testing of TNF specific antagonists. Another therapeutic approach being explored is the use of TNFR2 specific agonists, which could be administered alone or in combination with a TNFR1 antagonist.

An integrated cytokine and kynurenine network as the basis of neuroimmune communication.

Two of the molecular families closely associated with mediating communication between the brain and immune system are cytokines and the kynurenine metabolites of tryptophan. Both groups regulate neuron and glial activity in the central nervous system (CNS) and leukocyte function in the immune system, although neither group alone completely explains neuroimmune function, disease occurrence or severity. This essay suggests that the two families perform complementary functions generating an integrated network. The kynurenine pathway determines overall neuronal excitability and plasticity by modulating glutamate receptors and GPR35 activity across the CNS, and regulates general features of immune cell status, surveillance and tolerance which often involves the Aryl Hydrocarbon Receptor (AHR). Equally, cytokines and chemokines define and regulate specific populations of neurons, glia or immune system leukocytes, generating more specific responses within restricted CNS regions or leukocyte populations. In addition, as there is a much larger variety of these compounds, their homing properties enable the superimposition of dynamic variations of cell activity upon local, spatially limited, cell populations. This would in principle allow the targeting of potential treatments to restricted regions of the CNS. The proposed synergistic interface of 'tonic' kynurenine pathway affecting baseline activity and the superimposed 'phasic' cytokine system would constitute an integrated network explaining some features of neuroimmune communication. The concept would broaden the scope for the development of new treatments for disorders involving both the CNS and immune systems, with safer and more effective agents targeted to specific CNS regions.

Modulation of immune cell function, IDO expression and kynurenine production by the quorum sensor 2-heptyl-3-hydroxy-4-quinolone (PQS).

Many invasive micro-organisms produce 'quorum sensor' molecules which regulate colony expansion and may modulate host immune responses. We have examined the ability of Pseudomonas Quorum Sensor (PQS) to influence cytokine expression under conditions of inflammatory stress. The administration of PQS in vivo to mice with collagen-induced arthritis (CIA) increased the severity of disease. Blood and inflamed paws from treated mice had fewer regulatory T cells (Tregs) but normal numbers of Th17 cells. However, PQS (1µM) treatment of antigen-stimulated lymph node cells from collagen-immunised mice in vitro inhibited the differentiation of CD4+IFNγ+ cells, with less effect on CD4+IL-17+ cells and no change in CD4+FoxP3+Tregs. PQS also inhibited T cell activation by anti-CD3/anti-CD28 antibodies. PQS reduced murine macrophage polarisation and inhibited expression of IL1B and IL6 genes in murine macrophages and human THP-1 cells. In human monocyte-derived macrophages, IDO1 gene, protein and enzyme activity were all inhibited by exposure to PQS. TNF gene expression was inhibited in THP-1 cells but not murine macrophages, while LPS-induced TNF protein release was increased by high PQS concentrations. PQS is known to have iron scavenging activity and its suppression of cytokine release was abrogated by iron supplementation. Unexpectedly, PQS decreased the expression of indoleamine-2, 3-dioxygenase genes (IDO1 and IDO2), IDO1 protein expression and enzyme activity in mouse and human macrophages. This is consistent with evidence that IDO1 inhibition or deletion exacerbates arthritis, while kynurenine reduces its severity. It is suggested that the inhibition of IDO1 and cytokine expression may contribute to the quorum sensor and invasive actions of PQS.

Standardization of Antigen-Emulsion Preparations for the Induction of Autoimmune Disease Models.

Autoimmune murine disease models are vital tools for identifying novel targets and finding better treatments for human diseases. Complete Freund's adjuvant is commonly used to induce disease in autoimmune models, and the quality of the adjuvant/autoantigen emulsion is of critical importance in determining reproducibility. We have established an emulsification method using a standard homogenizer and specially designed receptacle. Emulsions are easy to prepare, form stable and uniform water-in-oil particles, are faster to make than the traditional syringe method, use less material and are designed to fill syringes with ease. In the present study, we have validated the emulsions for induction of experimental autoimmune encephalitis, collagen II induced arthritis, antigen induced arthritis, and delayed type hypersensitivity models. These models were induced consistently and reproducibly and, in some cases, the new method outperformed the traditional method. The method described herein is simple, cost-effective and will reduce variability, thereby requiring fewer animals for in vivo research involving animal models of autoimmune disease and in vaccine development.

Induction of IDO1 and Kynurenine by Serine Proteases Subtilisin, Prostate Specific Antigen, CD26 and HtrA: A New Form of Immunosuppression?

Several serine proteases have been linked to autoimmune disorders and tumour initiation although the mechanisms are not fully understood. Activation of the kynurenine pathway enzyme indoleamine-2,3-dioxygenase (IDO1) modulates cellular activity in the brain, tolerogenesis in the immune system and is a major checkpoint in cancer development. We now report that IDO1 mRNA and IDO1 protein expression (generating kynurenine) are induced in human monocyte-derived macrophages by several chymotryptic serine proteases with direct links to tumorigenesis, including Prostate Specific Antigen (PSA), CD26 (Dipeptidyl-peptidase-4, CD26/DPP-4), High Temperature Requirement protein-A (HtrA), and the bacterial virulence factor subtilisin. These proteases also induce expression of the pro-inflammatory cytokine genes IL1B and IL6. Other serine proteases tested: bacterial glu-C endopeptidase and mammalian Pro-protein Convertase Subtilase-Kexin-3 (PCSK3, furin), urokinase plasminogen activator (uPA), cathepsin G or neutrophil elastase, did not induce IDO1, indicating that the reported effects are not a general property of all serine proteases. The results represent a novel mechanism of activating immunosuppressive IDO1 and inducing kynurenine generation which, together with the production of inflammatory cytokines, would contribute to tumour initiation and progression, providing a new target for drug development. In addition, the proteasomal S20 serine protease inhibitor carfilzomib, used in the treatment of myeloma, prevented the induction of IDO1 and cytokine gene expression, potentially contributing to its clinical anti-cancer activity.

Disease status in human and experimental arthritis, and response to TNF blockade, is associated with MHC class II invariant chain (CD74) isoform expression.

Splice variants of CD74 differentially modulate the activity of cathepsin L (CTSL). As CD74 and CTSL participate in the pathogenesis of inflammatory diseases such as rheumatoid arthritis (RA), we determined whether splice variants of CD74 could be biomarkers of disease activity. Gene expression was measured in mice with collagen-induced arthritis using quantitative PCR (qPCR). In vitro studies using murine macrophage/DC-lineage cells determined the relative influence of macrophage phenotype on isoform expression and the potential to produce CTSL in response to TNF. CD74 splice variants were measured in human RA synovium and RA patients' monocytes. In arthritic mice, the expression of the p41 CD74 isoform was significantly higher in severely affected paws compared with unaffected paws or the paws of naïve mice; the p41 isoform significantly correlated with the expression of TNF in arthritic paws. Compared with M2-like macrophages, M1-like macrophages expressed increased levels of CD74 and had higher expression, secretion and activity of CTSL. RA patients that responded to TNF blockade had significantly higher expression levels of CD74 in circulating monocytes after treatment, compared with non-responders. The expression of the human CD74 isoform a was significantly higher in RA synovia, compared with osteoarthritis synovia, and was associated with CSTL enzymatic activity. This study is the first to demonstrate differential expression of the CD74 p41 isoform in an auto-immune disorder and in response to therapy. The differential expression of CD74 splice variants indicates an association, and potentially a mechanistic role, in the pathogenesis of RA.

Pharmacological modulation of T cell immunity results in long-term remission of autoimmune arthritis.

Chronic inflammatory diseases like rheumatoid arthritis are characterized by a deficit in fully functional regulatory T cells. DNA-methylation inhibitors have previously been shown to promote regulatory T cell responses and, in the present study, we evaluated their potential to ameliorate chronic and acute animal models of rheumatoid arthritis. Of the drugs tested, decitabine was the most effective, producing a sustained therapeutic effect that was dependent on indoleamine 2,3-dioxygenase (IDO) and was associated with expansion of induced regulatory T cells, particularly at the site of disease activity. Treatment with decitabine also caused apoptosis of Th1 and Th17 cells in active arthritis in a highly selective manner. The molecular basis for this selectivity was shown to be ENT1, a nucleoside transporter, which facilitates intracellular entry of the drug and is up-regulated on effector T cells during active arthritis. It was further shown that short-term treatment with decitabine resulted in the generation of a population of regulatory T cells that were able to suppress arthritis upon adoptive transfer. In summary, a therapeutic approach using an approved drug is described that treats active inflammatory disease effectively and generates robust regulatory T cells with the IDO-dependent capacity to maintain remission.

Rationale for Early Detection of EWSR1 Translocation-Associated Sarcoma Biomarkers in Liquid Biopsy.

Sarcomas are mesenchymal tumours that often arise and develop as a result of chromosomal translocations, and for several forms of sarcoma the EWSR1 gene is a frequent translocation partner. Sarcomas are a rare form of malignancy, which arguably have a proportionally greater societal burden that their prevalence would suggest, as they are more common in young people, with survivors prone to lifelong disability. For most forms of sarcoma, histological diagnosis is confirmed by molecular techniques such as FISH or RT-PCR. Surveillance after surgical excision, or ablation by radiation or chemotherapy, has remained relatively unchanged for decades, but recent developments in molecular biology have accelerated the progress towards routine analysis of liquid biopsies of peripheral blood. The potential to detect evidence of residual disease or metastasis in the blood has been demonstrated by several groups but remains unrealized as a routine diagnostic for relapse during remission, for disease monitoring during treatment, and for the detection of occult, residual disease at the end of therapy. An update is provided on research relevant to the improvement of the early detection of relapse in sarcomas with EWSR1-associated translocations, in the contexts of biology, diagnosis, and liquid biopsy.

TLR expression profiles are a function of disease status in rheumatoid arthritis and experimental arthritis.

The role of the innate immune system has been established in the initiation and perpetuation of inflammatory disease, but less attention has been paid to its role in the resolution of inflammation and return to homeostasis. Toll-like receptor (TLR) expression profiles were analysed in tissues with differing disease status in rheumatoid arthritis (RA), ankylosing spondylitis (AS), and in experimental arthritis. TLR gene expression was measured in whole blood and monocytes, before and after TNF blockade. In RA and osteoarthritis synovia, the expression of TLRs was quantified by standard curve qPCR. In addition, four distinct stages of disease were defined and validated in collagen-induced arthritis (CIA), the gold standard animal model for RA - pre-onset, early disease, late disease and immunised mice that were resistant to the development of disease. TLR expression was measured in spleens, lymph nodes, blood cells, liver and the paws (inflamed and unaffected). In RA whole blood, the expression of TLR1, 4 and 6 was significantly reduced by TNF blockade but the differences in TLR expression profiles between responders and non-responders were less pronounced than the differences between RA and AS patients. In RA non-responders, monocytes had greater TLR2 expression prior to therapy compared to responders. The expression of TLR1, 2, 4 and 8 was higher in RA synovium compared to control OA synovium. Circulating cytokine levels in CIA resistant mice were similar to naïve mice, but anti-collagen antibodies were similar to arthritic mice. Distinct profiles of inflammatory gene expression were mapped in paws and organs with differing disease status. TLR expression in arthritic paws tended to be similar in early and late disease, with TLR1 and 2 moderately higher in late disease. TLR expression in unaffected paws varied according to gene and disease status but was generally lower in resistant paws. Disease status-specific profiles of TLR expression were observed in spleens, lymph nodes, blood cells and the liver. Notably, TLR2 expression rose then fell in the transition from naïve to pre-onset to early arthritis. TLR gene expression profiles are strongly associated with disease status. In particular, increased expression in the blood precedes clinical manifestation.

TNFα inhibitors reduce bone loss in rheumatoid arthritis independent of clinical response by reducing osteoclast precursors and IL-20.

OBJECTIVES: About half of RA patients treated with TNFα inhibitors either do not respond or lose their initial therapeutic response over time. The clinical response is measured by reduction in DAS28, which primarily reflects inflammation. However, other effects of TNFα inhibitors, such as impact on bone erosion, are not assessed by DAS28. We aimed to examine the effect of TNFα inhibitors on bone density, bone biomarkers and cytokine production in responder and non-responder patients and assessed mechanisms of action. METHODS: BMD in the lumbar spine and femur neck of 117 RA patients was measured by DEXA scan. Bone turnover biomarkers CTX, osteoprotegerin (OPG), osteocalcin and RANKL were measured by ELISA. Levels of 16 cytokines in plasma and in tissue culture supernatants of ex vivo T cells were measured by multiplex assays and ELISA. The effect of treatment with TNFα inhibitors on blood mononuclear cell (MNC) differentiation to osteoclast precursors (OCP) was measured flow cytometry and microscopy. RESULTS: TNFα inhibitors improved lumbar spine BMD but had modest effects on blood bone biomarkers, irrespective of patients' clinical response. Blood OCP numbers and the ability of monocytes to differentiate to OCP in vitro declined after treatment. Treatment also reduced RANK expression and IL-20 production. BMD improvement correlated with reduced levels of IL-20 in responder patients. CONCLUSION: This study reveals that TNFα inhibitors reduce lumbar spine bone loss in RA patients irrespective of changes in DAS28. The reduction in bone loss is associated with reduction in IL-20 levels in responder patients.

IDO and Kynurenine Metabolites in Peripheral and CNS Disorders.

The importance of the kynurenine pathway in normal immune system function has led to an appreciation of its possible contribution to autoimmune disorders such as rheumatoid arthritis. Indoleamine-2,3-dioxygenase (IDO) activity exerts a protective function, limiting the severity of experimental arthritis, whereas deletion or inhibition exacerbates the symptoms. Other chronic disorder with an inflammatory component, such as atherosclerosis, are also suppressed by IDO activity. It is suggested that this overall anti-inflammatory activity is mediated by a change in the relative production or activity of Th17 and regulatory T cell populations. Kynurenines may play an anti-inflammatory role also in CNS disorders such as Huntington's disease, Alzheimer's disease and multiple sclerosis, in which signs of inflammation and neurodegeneration are involved. The possibility is discussed that in Huntington's disease kynurenines interact with other anti-inflammatory molecules such as Human Lymphocyte Antigen-G which may be relevant in other disorders. Kynurenine involvement may account for the protection afforded to animals with cerebral malaria and trypanosomiasis when they are treated with an inhibitor of kynurenine-3-monoxygenase (KMO). There is some evidence that changes in IL-10 may contribute to this protection and the relationship between kynurenines and IL-10 in arthritis and other inflammatory conditions should be explored. In addition, metabolites of kynurenine downstream of KMO, such as anthranilic acid and 3-hydroxy-anthranilic acid can influence inflammation, and the ratio of these compounds is a valuable biomarker of inflammatory status although the underlying molecular mechanisms of the changes require clarification. Hence it is essential that more effort be expended to identify their sites of action as potential targets for drug development. Finally, we discuss increasing awareness of the epigenetic regulation of IDO, for example by DNA methylation, a phenomenon which may explain differences between individuals in their susceptibility to arthritis and other inflammatory disorders.

IDO activation, inflammation and musculoskeletal disease.

The IDO/kynurenine pathway is now established as a major regulator of immune system function. The initial enzyme, indoleamine 2,3-dioxygenase (IDO1) is induced by IFNγ, while tryptophan-2,3-dioxygenase (TDO) is induced by corticosteroids. The pathway is therefore positioned to mediate the effects of systemic inflammation or stress-induced steroids on tissue function and its expression increases with age. Disorders of the musculoskeletal system are a common feature of ageing and many of these conditions are characterized by an inflammatory state. In inflammatory arthritis and related disorders, kynurenine protects against the development of disease, while inhibition or deletion of IDO1 increases its severity. The long-term regulation of autoimmune disorders may be influenced by the epigenetic modulation of kynurenine pathway genes, with recent data suggesting that methylation of IDO may be involved. Osteoporosis is also associated with abnormalities of the kynurenine pathway, reflected in an inversion of the ratio between blood levels of the metabolites anthranilic acid and 3-hydroxy-anthranilic acid. This review discusses evidence to date on the role of the IDO/kynurenine pathway and the highly prevalent age-related disorders of osteoporosis and rheumatoid arthritis and identifies key areas that require further research.

TNF receptor 2 signaling prevents DNA methylation at the Foxp3 promoter and prevents pathogenic conversion of regulatory T cells.

Regulatory T (Treg) cells expressing the transcription factor Foxp3 play an important role in maintaining immune homeostasis. Chronic inflammation is associated with reduced Foxp3 expression, function, and loss of phenotypic stability. Previous studies have established the importance of TNF receptor 2 (TNFR2) in the generation and/or activation of Treg cells. In this study, we assess the importance of TNFR2 in healthy mice and under inflammatory conditions. Our findings reveal that, in health, TNFR2 is important not only for the generation of Treg cells, but also for regulating their functional activity. We also show that TNFR2 maintains Foxp3 expression in Treg cells by restricting DNA methylation at the Foxp3 promoter. In inflammation, loss of TNFR2 results in increased severity and chronicity of experimental arthritis, reduced total numbers of Treg cells, reduced accumulation of Treg cells in inflamed joints, and loss of inhibitory activity. In addition, we demonstrate that, under inflammatory conditions, loss of TNFR2 causes Treg cells to adopt a proinflammatory Th17-like phenotype. It was concluded that TNFR2 signaling is required to enable Treg cells to promote resolution of inflammation and prevent them from undergoing dedifferentiation. Consequently, TNFR2-specific agonists or TNF1-specific antagonists may be useful in the treatment of autoimmune disease.

Functional and phenotypic heterogeneity of Th17 cells in health and disease.

BACKGROUND: Th17 cells have nonredundant roles in maintaining immunity, particularly at mucosal surfaces. These roles are achieved principally through the production of cytokines and the recruitment of other immune cells to maintain the integrity of mucosal barriers and prevent the dissemination of microorganisms. Th17 cells are heterogeneous and exhibit a considerable degree of plasticity. This allows these cells to respond to changing environmental challenges. However, Th17 cells also play pro-inflammatory roles in chronic autoimmune diseases. The trigger(s) that initiate these Th17 responses in chronic autoimmune diseases remain unclear. DESIGN: In this report, we provide an overview of studies involving animal models, patient data, genome wide association studies and clinical trials targeting IL-17 for treatment of patients to gain a better understanding of the pathogenic roles of Th17 cells play in a range of autoimmune diseases. RESULTS: The report sheds light on likely triggers that initiate or perpetuate Th17 responses that promote chronic inflammation and autoimmunity. The divergent effects of tumour necrosis factor alpha blockade on Th17 cells in patients, is explored. Furthermore, we highlight the role of Th17 cells in inducing autoreactive B cells, leading to autoantibody production. Pathogenic bacterial species can change Th17 cell phenotype and responses. These findings provide insights into how Th17 cells could be induced to promoting autoimmune disease pathogenesis. CONCLUSION: This article provides an overview of the distinct roles Th17 cells play in maintaining immunity at mucosal surfaces and in skin mucosa and how their functional flexibility could be linked with chronic inflammation in autoimmune rheumatic diseases.

Ibudilast Inhibits Chemokine Expression in Rheumatoid Arthritis Synovial Fibroblasts and Exhibits Immunomodulatory Activity in Experimental Arthritis.

OBJECTIVE: Ibudilast is a well-tolerated, orally available phosphodiesterase 4 (PDE4) inhibitor used to treat asthma and stroke. Since PDE4 inhibition suppresses inflammatory mediator production and cell proliferation in leukocytes, ibudilast may be a valuable therapy for the treatment of inflammatory autoimmune diseases such as rheumatoid arthritis (RA). This study was undertaken to assess the therapeutic potential of ibudilast by measuring its capacity to modulate inflammation in human leukocytes and RA synovial fibroblasts (RASFs) and in experimental arthritis. METHODS: Using standard curve quantitative polymerase chain reaction, the effect of ibudilast on gene expression in activated human leukocytes and RASFs was measured. Ibudilast was used to treat DBA/1 mice with collagen-induced arthritis, and an adoptive transfer model was used to assess its tolerogenic capacity. RESULTS: Ibudilast inhibited the expression of TNF, IL12A, and IL12B and the secretion of tumor necrosis factor (TNF) and interleukin-12 (IL-12)/23p40 from leukocytes, and reduced the expression of CCL5 and CCL3 in activated RASFs. Treatment of experimental arthritis with ibudilast resulted in a reduction in IL-17-producing cells and inhibition of disease progression. When combined with a TNF inhibitor, ibudilast caused marked suppression of active disease. Exposure of leukocytes from type II collagen-immunized DBA/1 mice to ibudilast in vitro attenuated their ability to adoptively transfer arthritis to DBA/1J-PrkdcSCID mice, providing evidence of an immunomodulatory effect. CONCLUSION: Our findings indicate that ibudilast reduces the expression and/or secretion of inflammatory mediators from activated human leukocytes and RASFs, inhibits Th17 cell responses in vivo, and improves established arthritis. Given the established safety profile of ibudilast in humans, its clinical evaluation in RA, either alone or in combination with a TNF inhibitor, should be considered.

Targeting early changes in the synovial microenvironment: a new class of immunomodulatory therapy?

OBJECTIVES: Controlled immune responses rely on integrated crosstalk between cells and their microenvironment. We investigated whether targeting proinflammatory signals from the extracellular matrix that persist during pathological inflammation provides a viable strategy to treat rheumatoid arthritis (RA). METHODS: Monoclonal antibodies recognising the fibrinogen-like globe (FBG) of tenascin-C were generated by phage display. Clones that neutralised FBG activation of toll-like receptor 4 (TLR4), without impacting pathogenic TLR4 activation, were epitope mapped by crystallography. Antibodies stained synovial biopsies of patients at different stages of RA development. Antibody efficacy in preventing RA synovial cell cytokine release, and in modulating collagen-induced arthritis in rats, was assessed. RESULTS: Tenascin-C is expressed early in the development of RA, even before disease diagnosis, with higher levels in the joints of people with synovitis who eventually developed RA than in people whose synovitis spontaneously resolved. Anti-FBG antibodies inhibited cytokine release by RA synovial cells and prevented disease progression and tissue destruction during collagen-induced arthritis. CONCLUSIONS: Early changes in the synovial microenvironment contribute to RA progression; blocking proinflammatory signals from the matrix can ameliorate experimental arthritis. These data highlight a new drug class that could offer early, disease-specific immune modulation in RA, without engendering global immune suppression.

TNFR signalling and its clinical implications.

Tumour necrosis factor-α (TNF-α) is a highly pleiotropic cytokine with effects on multiple pathological and physiological functions via two distinct receptors, TNFR1 and TNFR2. Much of the pro- inflammatory action of TNF-α is mediated by TNFR1 whereas TNFR2 is thought to play an immunoregulatory and tissue protective role. Anti-TNF- α biologics have been extremely successful in treating a number of immune mediated pathologies, including rheumatoid arthritis, ankylosing spondylitis, psoriasis, psoriatic arthritis and inflammatory bowel disease. However, anti-TNF therapy has been shown to induce systemic lupus erythematosus and psoriasis in some patients, and to be deleterious in multiple sclerosis. It is hypothesized that these paradoxical effects of anti-TNF-α are due to inhibition of TNFR2 signalling. In this review, we will focus on the biology and pathophysiologic role of TNF-α and on the therapeutic implications of targeting TNF-α receptor signalling.

T cell activation Rho GTPase activating protein (TAGAP) is upregulated in clinical and experimental arthritis.

Genome-wide association studies have identified various susceptibility variants and loci associated with incidence of rheumatoid arthritis (RA) in different populations. One of these is T cell activation Rho GTPase activating protein (TAGAP). The present study sought to measure the expression of TAGAP in RA patients, CD4+ T cells subsets from healthy humans and in mice with collagen-induced arthritis. Peripheral blood mononuclear cells (PBMC) from RA patients and tissues of arthritic mice at different stages of the disease were used for the evaluation of TAGAP mRNA expression. Increased TAGAP expression was observed in RA patients compared to healthy controls, and there were differences in the expression level of TAGAP in the tissues of mice with experimental arthritis. Gene expression in CD4+ T cells from healthy humans was greatest 4 h after activation and protein expression was greatest after 24 h. The expression of TAGAP was not correlated with CD4+ lymphocyte subsets which were enriched for functionally defined subsets (Th17, Treg, Th1), further indicating its utility as an indicator of lymphocyte activation. These findings indicate that increased TAGAP expression is a distinguishing feature of inflammatory disease and further highlight the role of TAGAP in RA susceptibility.

Response to Treatment with TNFα Inhibitors in Rheumatoid Arthritis Is Associated with High Levels of GM-CSF and GM-CSF+ T Lymphocytes.

Biologic TNFα inhibitors are a mainstay treatment option for patients with rheumatoid arthritis (RA) refractory to other treatment options. However, many patients either do not respond or relapse after initially responding to these agents. This study was carried out to identify biomarkers that can distinguish responder from non-responder patients before the initiation of treatment. The level of cytokines in plasma and those produced by ex vivo T cells, B cells and monocytes in 97 RA patients treated with biologic TNFα inhibitors was measured before treatment and after 1 and 3 months of treatment by multiplex analyses. The frequency of T cell subsets and intracellular cytokines were determined by flow cytometry. The results reveal that pre-treatment, T cells from patients who went on to respond to treatment with biologic anti-TNFα agents produced significantly more GM-CSF than non-responder patients. Furthermore, immune cells from responder patients produced higher levels of IL-1ß, TNFα and IL-6. Cytokine profiling in the blood of patients confirmed the association between high levels of GM-CSF and responsiveness to biologic anti-TNFα agents. Thus, high blood levels of GM-CSF pre-treatment had a positive predictive value of 87.5% (61.6 to 98.5% at 95% CI) in treated RA patients. The study also shows that cells from most anti-TNFα responder patients in the current cohort produced higher levels of GM-CSF and TNFα pre-treatment than non-responder patients. Findings from the current study and our previous observations that non-responsiveness to anti-TNFα is associated with high IL-17 levels suggest that the disease in responder and non-responder RA patients is likely to be driven/sustained by different inflammatory pathways. The use of biomarker signatures of distinct pro-inflammatory pathways could lead to evidence-based prescription of the most appropriate biological therapies for different RA patients.