Arthur G. Pratt

Mechanism of action of methotrexate in rheumatoid arthritis, and the search for biomarkers

The treatment and outcomes of patients with rheumatoid arthritis (RA) have been transformed over the past two decades. Low disease activity and remission are now frequently achieved, and this success is largely the result of the evolution of treatment paradigms and the introduction of new therapeutic agents. Despite the rapid pace of change, the most commonly used drug in RA remains methotrexate, which is considered the anchor drug for this condition. In this Review, we describe the known pharmacokinetic properties and putative mechanisms of action of methotrexate. Consideration of the pharmacodynamic perspective could inform the development of biomarkers of responsiveness to methotrexate, enabling therapy to be targeted to specific groups of patients. Such biomarkers could revolutionize the management of RA.

Current concepts in the pathogenesis of early rheumatoid arthritis

Rheumatoid arthritis (RA) is a systemic inflammatory disease with a predilection for symmetrically distributed diarthroidal joints. It is clinically heterogeneous, with particular disease phenotypes defined according to a complex interplay of genes and the environment. In this chapter we first summarize current knowledge of RA genetic susceptibility, a field which has been transformed in recent years by powerful modern genotyping technologies. The importance of a recently described subclassification for the disease based upon the presence or absence of circulating autoantibodies to citrullinated peptides has further informed genetic studies, and we consider the implications for our understanding of RA pathogenesis. We then review the cellular and molecular processes that initiate and perpetuate joint destruction.

Determination of thymic function directly from peripheral blood: a validated modification to an established method.

The thymus contributes naïve, self MHC reactive, self tolerant T cells to the peripheral immune system throughout life, albeit with a log-linear decline with age. Quantification of thymic function is clinically relevant in the setting of lymphoablation, but a phenotypic marker distinguishing recent thymic emigrants from long lived naïve T cells remains elusive. T cell receptor excision circles (TREC) are present in thymocytes exiting the thymus and quantification of the most frequent of these, the δrec-ψJα rearrangement has been widely used as a measure of recent thymic function. However, interpretation of results presented as TREC per cell has been criticised on the basis that extra-thymic cellular proliferation impacts on peripherally determined TREC numbers. TREC/ml is now considered to be more representative of thymic function than TREC/cell, especially where significant cellular proliferation occurs (e.g. during reconstitution following stem cell transplantation). Here we describe the validation of a novel variation to the established assay, directly quantifying TREC/ml from 300 µl whole blood. We show the assay to be reproducible, robust and stable longitudinally and we show equivalence of performance when compared with more standard assays. This assay particularly lends itself to the measurement of thymic function in children and where monitoring clinical variables is limited by tissue availability.

Synovial CD4+ T-cell-derived GM-CSF supports the differentiation of an inflammatory dendritic cell population in rheumatoid arthritis

Objective A population of synovial inflammatory dendritic cells (infDCs) has recently been identified in rheumatoid arthritis (RA) and is thought to be monocyte-derived. Here, we investigated the role and source of granulocyte macrophage-colony-stimulating factor (GM-CSF) in the differentiation of synovial infDC in RA. Methods Production of GM-CSF by peripheral blood (PB) and synovial fluid (SF) CD4+ T cells was assessed by ELISA and flow cytometry. In vitro CD4+ T-cell polarisation experiments were performed with T-cell activating CD2/CD3/CD28-coated beads in the absence or presence of pro-Th1 or pro-Th17 cytokines. CD1c+ DC and CD16+ macrophage subsets were flow-sorted and analysed morphologically and functionally (T-cell stimulatory/polarising capacity). Results RA-SF CD4+ T cells produced abundant GM-CSF upon stimulation and significantly more than RA-SF mononuclear cells depleted of CD4+ T cells. GM-CSF-producing T cells were significantly increased in RA-SF compared with non-RA inflammatory arthritis SF, active RA PB and healthy donor PB. GM-CSF-producing CD4+ T cells were expanded by Th1-promoting but not Th17-promoting conditions. Following coculture with RA-SF CD4+ T cells, but not healthy donor PB CD4+ T cells, a subpopulation of monocytes differentiated into CD1c+ infDC; a process dependent on GM-CSF. These infDC displayed potent alloproliferative capacity and enhanced GM-CSF, interleukin-17 and interferon-γ production by CD4+ T cells. InfDC with an identical phenotype to in vitro generated cells were significantly enriched in RA-SF compared with non-RA-SF/tissue/PB. Conclusions We demonstrate a therapeutically tractable feedback loop of GM-CSF secreted by RA synovial CD4+ T cells promoting the differentiation of infDC with potent capacity to induce GM-CSF-producing CD4+ T cells.

A CD4 T cell gene signature for early rheumatoid arthritis implicates interleukin 6-mediated STAT3 signalling, particularly in anti-citrullinated peptide antibody-negative disease

Objective We sought clinically relevant predictive biomarkers present in CD4 T-cells, or in serum, that identified those patients with undifferentiated arthritis (UA) who subsequently develop rheumatoid arthritis (RA). Methods Total RNA was isolated from highly purified peripheral blood CD4 T cells of 173 early arthritis clinic patients. Paired serum samples were also stored. Microarray analysis of RNA samples was performed and differential transcript expression among 111 ‘training cohort’ patients confirmed using real-time quantitative PCR. Machine learning approaches tested the utility of a classification model among an independent validation cohort presenting with UA (62 patients). Cytokine measurements were performed using a highly sensitive electrochemiluminescence detection system. Results A 12-gene transcriptional ‘signature’ identified RA patients in the training cohort and predicted the subsequent development of RA among UA patients in the validation cohort (sensitivity 68%, specificity 70%). STAT3-inducible genes were over-represented in the signature, particularly in anti-citrullinated peptide antibody-negative disease, providing a risk metric of similar predictive value to the Leiden score in seronegative UA (sensitivity 85%, specificity 75%). Baseline levels of serum interleukin 6 (IL-6) (which signals via STAT3) were highest in anti-citrullinated peptide antibodies-negative RA and distinguished this subgroup from non-RA inflammatory synovitis (corrected p<0.05).Paired serum IL-6 measurements correlated strongly with STAT3-inducible gene expression. Conclusion The authors have identified IL-6-mediated STAT-3 signalling in CD4 T cells during the earliest clinical phase of RA, which is most prominent in seronegative disease. While highlighting potential biomarker(s) for early RA, the role of this pathway in disease pathogenesis awaits clarification.

IL-6-driven STAT signalling in circulating CD4+ lymphocytes is a marker for early anticitrullinated peptide antibody-negative rheumatoid arthritis

Objectives A previously identified signal transduction and activator of transcription-3 (STAT3) target-enriched gene signature in circulating CD4+ T cells of patients with early rheumatoid arthritis (RA) was prominent in autoantibody-negative individuals. Here, interleukin (IL)-6-mediated STAT signalling was investigated in circulating lymphocytes of an independent early arthritis patient cohort, seeking further insight into RA pathogenesis and biomarkers of potential clinical utility. Methods Constitutive and IL-6-induced expression of phosphorylated STAT1 (pSTAT1) and pSTAT3 was determined in T and B cells using Phosflow cytometric analysis in patients with RA and controls. Contemporaneous levels of serum cytokines were measured by immunoassay. Induced gene expression was measured in cultured CD4+T cells by quantitative real-time PCR. Results Among circulating lymphocytes of 187 patients with early arthritis, constitutive pSTAT3 correlated with serum IL-6 levels maximally in CD4+ T cells. Increased constitutive pSTAT3, but not pSTAT1, was observed in circulating CD4+ T cells of patients with early anticitrullinated peptide autoantibody (ACPA)-negative RA compared with disease controls, and these levels decreased alongside markers of disease activity with IL-6R-targeted treatment. Among patients presenting with seronegative undifferentiated arthritis (UA) the ratio of constitutive pSTAT3:pSTAT1 in CD4+ T cells contributed substantially to an algorithm for predicting progression to classifiable RA during a median of 20 months follow-up (area under receiver operator characteristic curve=0.84; p<0.001). Conclusions Our findings support a particular role for IL-6-driven CD4+ T cell activation via STAT3 during the induction of RA, particularly as a feature of ACPA-negative disease. CD4+ T cell pSTAT measurements show promise as biomarkers of UA–RA progression and now require independent validation.

Synovial tissue research: a state-of-the-art review

The synovium is the major target tissue of inflammatory arthritides such as rheumatoid arthritis. The study of synovial tissue has advanced considerably throughout the past few decades from arthroplasty and blind needle biopsy to the use of arthroscopic and ultrasonographic technologies that enable easier visualization and improve the reliability of synovial biopsies. Rapid progress has been made in using synovial tissue to study disease pathogenesis, to stratify patients, to discover biomarkers and novel targets, and to validate therapies, and this progress has been facilitated by increasingly diverse and sophisticated analytical and technological approaches. In this Review, we describe these approaches, and summarize how their use in synovial tissue research has improved our understanding of rheumatoid arthritis and identified candidate biomarkers that could be used in disease diagnosis and stratification, as well as in predicting disease course and treatment response.

Profound invariant natural killer T-cell deficiency in inflammatory arthritis

Objectives Data from rodent models indicate that invariant natural killer T (iNKT) cells are key regulators of many immune responses including autoimmune arthritis, but their role in human diseases is unclear. The aims of this study are to determine whether iNKT cell frequency and function are altered in patients with rheumatoid arthritis (RA), and the clinical significance of such iNKT cell abnormalities. Methods Peripheral blood iNKT cell frequency and proliferative response to an iNKT cell-specific agonist, α-galactosylceramide were measured in 46 RA patients (including 23 untreated, newly diagnosed patients), 22 healthy controls and 27 patients presenting with recent-onset joint pain. The relationship between iNKT cell frequency and clinical characteristics and the effects of immunosuppressive treatment was examined. Results Compared with healthy controls, RA patients had a decreased frequency of peripheral blood iNKT cells (median 0.001% vs 0.021%, p<0.001) and the proliferative response of this subset to α-galactosylceramide was also diminished in the patient group (median fold-expansion 31 vs 121, p=0.037). These abnormalities preceded the initiation of disease-modifying or immunosuppressive therapy, whose effect was to increase the circulating iNKT cell frequency (p=0.037). Furthermore, iNKT cell frequency correlated inversely with the systemic inflammatory marker, C-reactive protein (p=0.008). Finally, in patients presenting with recent-onset joint symptoms, normal peripheral blood iNKT cell frequency predicted a non-inflammatory cause of joint pain. Conclusion iNKT cell deficiency is present in patients with RA and other inflammatory arthropathy. Normal iNKT cell frequency predicts non-inflammatory causes of joint pain.

Tolerogenic dendritic cells generated with dexamethasone and vitamin D3 regulate rheumatoid arthritis CD4+ T cells partly via transforming growth factor-β1

Tolerogenic dendritic cells (tolDC) are a new immunotherapeutic tool for the treatment of rheumatoid arthritis (RA) and other autoimmune disorders. We have established a method to generate stable tolDC by pharmacological modulation of human monocyte‐derived DC. These tolDC exert potent pro‐tolerogenic actions on CD4+ T cells. Lack of interleukin (IL)−12p70 production is a key immunoregulatory attribute of tolDC but does not explain their action fully. Here we show that tolDC express transforming growth factor (TGF)‐β1 at both mRNA and protein levels, and that expression of this immunoregulatory cytokine is significantly higher in tolDC than in mature monocyte‐derived DC. By inhibiting TGF‐β1 signalling we demonstrate that tolDC regulate CD4+ T cell responses in a manner that is at least partly dependent upon this cytokine. Crucially, we also show that while there is no significant difference in expression of TGF‐βRII on CD4+ T cells from RA patients and healthy controls, RA patient CD4+ T cells are measurably less responsive to TGF‐β1 than healthy control CD4+ T cells [reduced TGF‐β‐induced mothers against decapentaplegic homologue (Smad)2/3 phosphorylation, forkhead box protein 3 (FoxP3) expression and suppression of (IFN)‐γ secretion]. However, CD4+ T cells from RA patients can, nonetheless, be regulated efficiently by tolDC in a TGF‐β1‐dependent manner. This work is important for the design and development of future studies investigating the potential use of tolDC as a novel immunotherapy for the treatment of RA.

Tolerogenic dendritic cells generated with dexamethasone and vitamin D3 regulate rheumatoid arthritis CD4(+) T-cells partly via TGF-β1.

Tolerogenic dendritic cells (tolDC) are a new immunotherapeutic tool for the treatment of rheumatoid arthritis (RA) and other autoimmune disorders. We have established a method to generate stable tolDC by pharmacological modulation of human monocyte‐derived DC. These tolDC exert potent pro‐tolerogenic actions on CD4+ T cells. Lack of interleukin (IL)−12p70 production is a key immunoregulatory attribute of tolDC but does not explain their action fully. Here we show that tolDC express transforming growth factor (TGF)‐β1 at both mRNA and protein levels, and that expression of this immunoregulatory cytokine is significantly higher in tolDC than in mature monocyte‐derived DC. By inhibiting TGF‐β1 signalling we demonstrate that tolDC regulate CD4+ T cell responses in a manner that is at least partly dependent upon this cytokine. Crucially, we also show that while there is no significant difference in expression of TGF‐βRII on CD4+ T cells from RA patients and healthy controls, RA patient CD4+ T cells are measurably less responsive to TGF‐β1 than healthy control CD4+ T cells [reduced TGF‐β‐induced mothers against decapentaplegic homologue (Smad)2/3 phosphorylation, forkhead box protein 3 (FoxP3) expression and suppression of (IFN)‐γ secretion]. However, CD4+ T cells from RA patients can, nonetheless, be regulated efficiently by tolDC in a TGF‐β1‐dependent manner. This work is important for the design and development of future studies investigating the potential use of tolDC as a novel immunotherapy for the treatment of RA.