Association of CD39 expression on Treg cells with the therapeutic efficacy of methotrexate in treatment of rheumatoid arthritis

Abstract

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease that affects 1% of world population and is characterized by an infiltration of immune cells in the joint. The most commonly used therapeutic approach for treating RA is the use of low doses of Methotrexate (MTX), a diidrofolate reductase inhibitor whose anti-inflammatory effect in RA is associated with its capacity in promoting the maintenance of elevated levels of adenosine (ADO) extracellular, by a mechanism dependent on the activity of ATP degradation exerted by ectonucleotidases and CD39/ENTPD1 CD73/ecto-5 'nucleotidase. However, a considerable proportion of patients are refractory to treatment with MTX and the mechanism by which this phenomenon occurs is not yet established. In a recent study from our research group, we observed that patients with RA not responsive to MTX treatment (UR-MTX) have a lower CD39 expression on regulatory T cells (Tregs) circulating, cell subtype described bypresenting a high ectoenzimes expression on their surface, which causes a reduced ability to produce extracellular ADO by these cells. Therefore, the analysis of changes in the process of regulating CD39 expression on Treg cells is one of the first steps to clarify the phenomena related to MTX unresponsiveness. Although there are no reports of the induction mechanism of CD39 expression on Tregs cells, the literature reports that induction of CD39 expression is related to CREB (connecting element to respond to cAMP) activation, which occurs through a signal dependent on protein kinase A (PKA) activity or the intracellular transducer signal, SMADs. On Treg cells, CREB activation signaling is induced by TGF-², a cytokine crucial to differentiation process of this cellular subtype. Based on this information, it is plausible that CD39 expression on Treg cells is induced by TGF-², which acts by activating CREB-dependent signaling via SMAD / CREB or PKA / CREB. In addition, it is likely that there is a change in this signaling pathway in patients UR-MTX. So, the aim of this study is to identify the mechanism of induction of CD39 expression on Tregs cells, and to evaluate whether MTX unresponsiveness is based on changes in these molecular pathways on Tregs cells. We propose: a) to demonstrate the mechanism of induction and regulation of CD39 expression on Tregs cells; b) to identify changes on expression and activation of molecules related to TGF-b signaling in inducing CD39 expression in UR-MTX patients; c) to evaluate the therapeutic potential of Apyrase (CD39 purified) in the development of arthritis in experimental model of antigen-induced arthritis (AIA). (AU)