Research Interests

Until recently, the research interests of my group had been centred around the molecular event that trigger the development of autoimmune diseases. Rheumatoid arthritis (RA) is a chronic inflammatory disease of the synovium, whose pathogenesis is uncertain. A number of features suggest an autoimmune disease although there are competing theories. RA T-cells are hypo-responsive to stimulation and RA patients are clinically immuno-suppressed. Specific abnormalities within the naïve T-cell compartment (repertoire contraction and shortening of telomeres) together with reports of unusual mature T-cell expansions have suggested a possible defect in the generation and/or the maintenance of T-cells in RA. During the past 7 years, I have focussed on identifying potential primary defects in T-cell development (thymic activity, 1 2 3 ) and maturation (T-cell differentiation 1 4 ), the activity of CD4+CD25high regulatory T cells 5 , certain signalling pathways involved in anergy, activation and T-cell regulation (Jagged/Notch 6 7 8 ) and more recently the role of IL-7 in RA 3 . These projects are ongoing and will be further developed in the future. I am currently expanding upon this work further and developing new projects in relation to the molecular events leading to the establishment of RA. Over the last 12 months I however, developed an interest in Mesenchymal Stem cell, as they are the source of IL-7 secretion in several tissues. This has recently been recognised by the award of an academic Senior Researcher fellowship.

Previous work

Thymic activity measurements. The development of techniques for measuring T-cell receptor excision circles (TRECs) has recently provided a surrogate measure of thymic activity. Each mature T-cell exiting the thymus contains episomal TRECs derived from rearranged alpha and beta gene loci. As these naïve cells expand in the periphery, TRECs do not replicate but are distributed among daughter cells are thus gradually diluted in the peripheral T-cell pool. Therefore, by measuring the proportion of peripheral T-cells containing TRECs 2 , an estimate of thymic function can be obtained. However, a number of dynamic processes influence TREC measurements, and under normal homeostatic conditions, we have shown that variations in the TREC-content are more influenced by peripheral T-cell proliferation than by variation of the thymic output 1 . Nevertheless, following therapeutically induced lymphopenia, T-cell counts and TREC levels are low, and therefore a subsequent accumulation reflects true thymic output 3 .

Markers of T-cell differentiation. A major limitation in addressing T-cell differentiation abnormalities in RA has been a relatively poor understanding of normal T-cell differentiation, and limited characterisation of phenotypic differentiation markers. Thus, isoforms of the tyrosine phosphatase CD45 (RB, RA and RO), along with expression of the lymph node homing receptor CD62L are frequently used to distinguish “naïve” from “memory” T-cells. We used these markers in conjunction with TREC measurements to define a model of T-cell differentiation. 1,3 .

Thymic activity and T-cell differentiation are abnormal in RA. Thymic function and T-cell differentiation were analysed in a cross-section of RA patients, focusing particularly on early disease. We demonstrated a significant perturbation of T-cell dynamics in RA 1 A smaller proportion of CD4+ and CD8+ T-cells contained a TREC, and the normal relationship between TREC-content and age was lost. Furthermore, RA patients had fewer naïve T-cells than healthy controls, but instead possessed T-cells of various atypical phenotypes. These included immature cells that co-expressed naïve and memory markers. These cells were hyper-responsive to mitogenic or TCR stimulation. Levels of inflammation correlated positively with the size of the immature atypical subsets 1 .

Absence of T-cell reconstitution following therapeutic lymphodepletion. A prolonged CD4+T-cell lymphopenia is observed in RA patients after lymphodepleting therapy 3 . We documented the thymic response to lymphopenia in RA and cancer patients undergoing high-dose cytotoxic therapy with Autologous Stem Cell Transplantation (ASCT) 3 . The critical difference between RA and cancer patients was the inability of RA T-cells to expand in response to lymphopenia. This seemed to be the major factor limiting reconstitution. A cytokine involved in the survival of pre-B and T-cell progenitor in the bone marrow, T-cell development in the thymus and T-cell function in the periphery, is IL-7. This cytokine is also a major factor in inducing peripheral T-cell expansion in response to lymphopenia. We analysed the levels of circulating Il-7 in RA and cancer patients following ASCT, and preliminary data, showed no increase in RA as compared to 5-fold rises in cancer.

Molecular events leading to the establishment of RA . One of the major issues in rheumatology is the early diagnostic of RA, which determines the chances of controlling efficiently this disease. It has been established clinically that a treatment has more chances of success if it can be administered within a very short window of about 12 to 15 weeks from the development of initial symptoms. During this time a number of events will determine if the disease will persist and become chronic or will resolve. A T-cell survival factor, produced locally in the synovium by stromal cells, may determine the persistence of a T-cell response and induce T-cell independent–chronic inflammation. The identity of this factor is not known. Following from a differential expression array between persistent and self-limiting disease 7 an interesting set of genes are under investigation.

IL-7 deficiency in RA . The absence of IL-7 response to lymphopenia suggested a possible defect in regulating the expression of this cytokine. We measured circulating levels of IL-7 in a cross section of active RA patients, RA patients in clinical remission, osteoarthritis patients (OA) and healthy controls. IL-7 levels are lower in active RA, but not in OA, compared to controls 3 . Levels in remission are heterogeneous ranging from low to normal levels. IL-7 in patients in remission is positively correlated with TREC levels confirming the effect of IL-7 on thymic activity 3 .

Very little is known about the regulation of IL-7 expression in humans. Although IL-7 circulates in the periphery and is modulated, its origin remains unclear. IL-7 expression is induced by IFN- g whereas TGF- b is a negative regulator. In the synovial membrane, IL-7 expression can be induced in stromal cells, by TNF- a and IL-1 where it is believed to trigger the expression of RANKL and GM-CSF in T-cells and ultimately induces the differentiation of osteoclasts. TNF-blockade therapy in vivo had however, no significant effect on IL-7 3 . Therefore, it is possible that similar stimuli will have different effect on IL-7 expression according to the tissues involved. It is most likely that the microenvironment has a major contribution on controlling the effect that these cytokines have on each other's expression.

The Jagged / Notch pathway. The Jagged / Notch pathway is a membrane bound ligand/receptor system. Engagement of the Notch receptor by Jagged or Delta induces the release of the internal domain of Notch, which can then signal via two known pathways. The first pathway leads to de-repression of a number of target genes for specific transcription factors, such as HES-1, Deltex, Nur77 or NF k B. The second pathway involves the release of Grb2 (inhibitor of tyrosine kinases) from a Deltex/Grb2-blocking complex. Many animal models have shown the importance of this pathway in bone marrow progenitor development, thymic T-cell selection, in the generation of peripheral tolerance, in the induction of anergy 6 and in the mediation of immune regulation by CD4+/CD25+ T-cells 5 . The expression of the different members of the Jagged / Notch pathway is abnormal in RA, and these abnormalities are present at the earlier stages of the disease 8 . Signalling through the Notch receptor has been demonstrated in RA but not in osteo-arthritis, and TNF- a modulates Notch signalling in RA fibroblasts to induce their proliferation. Interestingly, the expression of Notch 4 is reduced in RA patients in clinical remission, but the expression of Jagged 1, Notch 1 and Delta 1 remains high 9 Notch 4 over-expression is a feature of anergic T-cells 6 and activated healthy CD4+ CD25+ regulatory T-cells 5 . It is therefore possible that modifications in the basal levels of expression of these proteins in RA may well favour the selection of auto-reactive T-cells, the development of anergy and/or the breakdown of tolerance/regulation.

More recent work

Mesenchymal stem cell differentiation (i n collaboration with Prof D. McGonagle and Dr E. Jones, Rheumatology Research, Mesenchymal stem cell biology group) . Dr Jones has recently been able to isolate, grow and differentiate Mesenchymal progenitor stem cells (MSCs) from the bone marrow of healthy individuals and patients with RA 10 . These cells have the ability to differentiate into bone, cartilage, muscle or fat tissue, in addition to their normal function in supporting haematopoiesis in the bone marrow. There is a circumstantial evidence however that this pluripotency is lost or diminished in RA. Similar cells can also be isolated from different tissues, and particularly from the synovial fluid of patients with different form of arthropathies: osteoarthritis (OA), reactive arthritis (ReA) or RA 11 . Our preliminary evidence suggests that cells isolated from these disease states also have differing capabilities, RA cells are less osteogenic and OA cells more prone to develop into cartilage. In order to understand how the microenvironment can influence the fate of MSCs, we have developed assays both to quantify the differentiation at the cellular level and to analyse the expression of a number of markers for each differentiation pathways. However, due to the very limited amount of clinical material available, it has been imperative to use real-time PCR quantification for the latter purpose. We use, for example, SOX9 as a marker of cartilage differentiation, RUNX2 for bone, IL7 for haematopoietic support, MYOD for muscle and PPAR g for fat. We have shown that these genes are specifically up-regulated during the corresponding process of differentiation from healthy MSCs. We have also shown differences in the level of expression of these genes in MSCs (synovial fluid cells) from different arthritic diseases. Using this information, we are now in a position to address in detail how these genes are regulated under different inflammatory conditions. This will permit assessment of the role of individual patterns of stem cell differentiation in disease pathogenesis and in the repair of joint damage.

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Group leader : Pathogenesis of Immune Mediated Inflammatory Diseases (IMID).

Head of the real time PCR centre

Head of the translational Research centre for Rheumatology.