DCS · Dynamic regulation of cytokine signalling in lymphocytes during inflammation

Dynamic regulation of cytokine signalling in lymphocytes during inflammation

Address of the project CIG is developed at the Immune synapse and Intracellular signalling group of the Department of Microbiology I (Immunology) at the Faculty of Medicine of the University Complutense of Madrid (UCM), Spain (https://www.ucm.es/microbiologia-1/ISIS). Overview of results and conclusions Human protein tyrosine phosphatases (PTPs) constitute a family of enzymes encoded by 107 genes that regulate phospho-tyrosine levels. Hematopoietic cells express around 70% of these genes. Thus, it is expected that a coordinated function of PTPs is needed, especially of those enzymes that share the same substrate. In T cells, PTPs act in signalling networks organised downstream many different surface receptors, including antigen receptors, Integrins, co-stimulatory molecules, and cytokine receptors. The immunological synapse (IS) provides a spatial frame used by lymphocytes to sense extracellular stimuli received by all these receptors. IS-polarised signalling networks and the cell machinery (including cytoskeleton and endosomal compartment) integrate extracellular information and thereby control the cell response. We are studying the molecular dynamics of IS-polarised signalling components at the IS, and the role of PTPs not only in this molecular dynamics but also in the lymphocyte activation and function. We have also compared the expression of genes coding for PTPs in CD4 T cells of healthy donors and rheumatoid arthritis (RA) patient, as a model of autoimmunity. The following research lines have been/are being developed: 1. We have determined the expression profile of PTPs and suppressors of cytokine signalling (SOCS) in CD4+ T cells of rheumatoid arthritis (RA) diseased individuals and healthy donors. CD4+ T cells are thought to be involved in RA, particularly in anti-citrullinated peptide antibody positive (ACPA+) patients. We have found changes in expression levels of certain genes in RA patients, including the dual specific phosphatase DUSP7 and the cell cycle regulator CDC25B. Low expression level of DUSP7 was restricted to patients with positive rheumatoid factor and ACPA. The altered expression of CDC25B correlated with the activity of the disease. Thus, the lower expression of this phosphatase might have a functional impact in the T cell immune response leading to the progression of the disease. The possible role of these PTPs as diagnostic or activity biomarkers in clinical protocols will be further investigated (Castro-Sanchez P. et al. Clinical Exp Immunol, under review). 2. A systematic analysis of the expression profile of PTPs has also been carried out during Th1-polarising conditions and upon PKC activation and intracellular raise of Ca2+ of effector cells. Changes in gene expression levels suggest a previously non-noted regulatory role of several PTPs in Th1 polarisation and effector function. A substantial change in the spatial compartmentalisation of ERK activity during T cell responses is proposed based on changes in the dose of cytoplasmic and nuclear MAPK phosphatases (MKPs). This study also suggests a regulatory role of autoimmune-related PTPs in controlling T helper polarisation in humans. We expect that those PTPs that regulate T helper polarisation will constitute potential targets for intervening CD4 T cell immune responses in order to generate new therapies for the treatment of autoimmune diseases (Castro-Sanchez P. et al. J Immunol Res, Under review). 3. We have studied the role of JAKs (Janus Kinases) in the assembly of the IS. Our data indicate that JAKs are not needed for IS assembly, as assessed for the polarisation of actin cytoskeleton and talin, as well as for the induction of phosphor-tyrosine at the contact site between T cells and antigen presenting cells. 4. We have set-up the routines to develop fluorescence correlation and cross-correlation spectroscopy (FCS and FCCS) experiments. These methods enable us to measure the diffusion coefficient of molecules organised in signalling complexes. By this approach we have identified and characterised two pools, one cytosolic and one membrane, of the phosphatase of regenerating liver-1 (PRL-1) in T cells. The intracellular dynamics and the function of this molecule during T cell activation have been studied (Castro-Sanchez P. et al. Manuscript in preparation). The function of PRL-3 in the molecular dynamics at the IS and in the T cell activation is being currently studied. 5. We have analysed the regulation of the signalling module for cofilin activation in T cells. Based on the regulatory role of the phosphatase Slingshot-1 (SSH1) in cofilin activation, an ultrasensitive response of the module has been proposed. We hypothesize that ultrasensitivity in the cofilin signalling module might tune immune responses by T cells (Ramirez-Muñoz et al. Front Immunol 2016). The function of SSH1 in the molecular dynamics at the IS and in the T cell activation is being currently studied. Potential socio-economic impact We expect that our project will have a clear impact in biomedicine and, consequently, the developed research will be relevant for civil society. Understanding how signalling networks are regulated in immune cells will contribute to the design of new therapeutic strategies for human autoimmune diseases. Our project is advancing on 3 aspects to be consider: - Expression patterns associated to autoimmune diseases might help on diagnostic, prognosis, or estimation of the activity of the disease. In this regard expression levels of CDC25B might be a good biomarker for RA disease activity. - Expression studies help to identify new regulators of T cell activation. Several PTPs have been found clearly regulated upon Th1 polarising conditions, suggesting a potential role of them during polarisation. These data will constitute new potential therapeutic targets. - We expect that our project will help to understand how the molecular dynamics critical for T cell responses is regulated and altered in pathological scenarios. Manipulating pathology-associated molecular dynamics will then offer new avenues for therapy. Research career development and re-/ integration of the fellow The Department of Microbiology I (Immunology) of the University Complutense of Madrid (I-UCM) has provided me with all the independence to start my own research group (https://www.ucm.es/microbiologia-1/ISIS) and currently I have 2 PhD students, that will finish their thesis by the end of this year. We continuously expand the group with yearly-based coming Master students, which some of them will be encouraged to stay with us. I-UCM has allowed me to teach full theoretical and practical lectures in the different University grades and Master studies. Experience in teaching and research activities have helped me to pass the needed evaluations to obtain an associate professor position at the I-UCM. In this regard, the UCM has a professional track for providing ‘Ramon y Cajal’ (RyC) researchers with a stable permanent position. The CIG has been essential to get this integration in the Spanish science system.

Data: CORDIS, © European Union