Project

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Purinergic control of adaptive immunity by P2X7 receptor

Applicant Grassi Fabio
Number 140963
Funding scheme Project funding
Research institution Istituto di ricerca in biomedicina (IRB) Facoltà di scienze biomedice
Institution of higher education Università della Svizzera italiana - USI
Main discipline Immunology, Immunopathology
Start/End 01.04.2012 - 31.03.2015
Approved amount 495'000.00
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Keywords (6)

Purinergic signaling; Signal transduction; Autoimmunity; T cell; P2X receptors; Adaptive immunity

Lay Summary (English)

Lead
Lay summary
The present project aims at investigating the role played by extracellular adenosine 5’-triphosphate (ATP) in regulating the function of a subset of white blood cells, namely T lymphocytes. ATP is usually confined inside the cell where it constitutes the source of chemical energy for the majority of cellular functions. However, ATP has also a less widely appreciated function as signaling molecule in the extracellular space. In fact, ATP can be released by mammalian cells and activate so called purinergic receptors in the plasma membrane, known as P2. Two classes of P2 receptors exist in eukaryotic cells. The first is constituted by P2Y receptors, which are metabotropic, i.e. coupled to heterotrimeric guanine nucleotide-binding protein (G-protein) and modulate mainly intracellular calcium as well as cyclic AMP levels; the second is composed by P2X receptors, which are ionotropic, i.e. ligand-gated cation-permeable channels that open when bound to ATP. A wealth of functions is regulated by P2 receptors, including secretion, tissue blood flow, cell volume and inflammation. In fact, ATP is massively released upon cell death and acts as a “danger associated molecular pattern” (DAMP) for cells of the innate immune system. P2X7 is the principal P2X subtype expressed in T lymphocytes. We have recently demonstrated that ATP inhibits the development and function of immunosuppressive regulatory T cells (Tregs) through P2X7 receptor stimulation. This T cell subset is of fundamental importance in maintaining the integrity of our organism since its defect results in severe autoimmune manifestations with tissue destruction in multiple organs. The present project is aimed at characterizing signal transduction events elicited by P2X7 stimulation in T cells, identifying critical molecules in this signalling cascade and defining the role of P2X7 in small intestine mucosa, a site where ATP is found in the extracellular space and bacteria constitute a continuous challenge for cells of the immune system associated with mucosa. In this respect we will investigate possible differences in gut microflora in mice in which T cells are devoid of P2X7 receptor because of a deletion in the gene coding for P2X7. The experiments proposed in this project will provide insights into the role of P2X7-mediated regulation of T cell function and adaptive immune response. Given that basic science is a fundamental step towards translational research, understanding the molecular mechanisms that govern T cell responsiveness will improve our knowledge of the pathogenesis of autoimmune diseases. The project is expected to delineate novel targets amenable to pharmacological intervention in autoimmune disorders. Epidemiological relevance of autoimmune diseases in modern societies is such that the discovery of therapeutic targets is a major priority in the setting of health research policies.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Ectonucleotidase activity and immunosuppression in astrocyte-CD4 T cell bidirectional signaling.
Filipello Fabia, Pozzi Davide, Proietti Michele, Romagnani Andrea, Mazzitelli Sonia, Matteoli Michela, Verderio Claudia, Grassi Fabio (2016), Ectonucleotidase activity and immunosuppression in astrocyte-CD4 T cell bidirectional signaling., in Oncotarget, 7(5), 5143-56.
Enhancement of muscle T regulatory cells and improvement of muscular dystrophic process in mdx mice by blockade of extracellular ATP/P2X axis
Gazzerro Elisabetta, Bruno Claudio, Grassi Fabio (2015), Enhancement of muscle T regulatory cells and improvement of muscular dystrophic process in mdx mice by blockade of extracellular ATP/P2X axis, in The American Journal of Pathology, 185, 3349-3360.
ATP-Gated Ionotropic P2X7 Receptor Controls Follicular T Helper Cell Numbers in Peyer’s Patches to Promote Host-Microbiota Mutualism
Proietti Michele (2014), ATP-Gated Ionotropic P2X7 Receptor Controls Follicular T Helper Cell Numbers in Peyer’s Patches to Promote Host-Microbiota Mutualism, in Immunity, 41, 789-801.
Dependence of Immunoglobulin Class Switch Recombination in B Cells on Vesicular Release of ATP and CD73 Ectonucleotidase Activity
Schena Francesca (2013), Dependence of Immunoglobulin Class Switch Recombination in B Cells on Vesicular Release of ATP and CD73 Ectonucleotidase Activity, in Cell Reports, 3, 1824-1831.

Collaboration

Group / person Country
Types of collaboration
Dr. Elisabetta Traggiai, Novartis Institute for Research in Biomedicine Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Dr. Claudia Verderio, Institute of Neuroscience, CNR, Milan Italy (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Dr. Claudio Bruno, Istituto Gaslini, Genoa Italy (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Exchange of personnel
Dr. Kathy D. McCoy, UVCM, University of Bern Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. Gabriella Tedeschi, University of Milan Italy (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Exchange of personnel
Dr. Emma Slack, Institute of Microbiology, ETH Zurich Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
Meeting of the Swiss Immunology PhD students Talk given at a conference Dependance of Immunoglobulin CSR in B cells on vesicular release of ATP and CD73 ectonucleotidase activity 14.04.2015 Wolfsberg, Switzerland Faliti Caterina Elisa;
Keystone Symposium "Gut Microbiota Modulation of Host Physiology: The Search for Mechanism" Poster Altered microbiota by enhanced T follicular help and hyper IgA results in impaired glucose metabolism 01.03.2015 Keystone (CO), United States of America Perruzza Lisa;
XXIV AINI Congress Talk given at a conference Astrocyte-T cell cross-talk 01.10.2014 Sorrento, Italy Grassi Fabio;
18th Germinal Centre Conference Talk given at a conference Dependance of Immunoglobulin CSR in B cells on vesicular release of ATP and CD73 ectonucleotidase activity 11.09.2014 Uddevalla, Sweden Faliti Caterina Elisa;
18th Germinal Centre Conference Poster Altered microbiota by enhanced T Follicular Help and hyper IgA results in impaired glucose metabolism 11.09.2014 Uddevalla, Sweden Perruzza Lisa;
9th FENS Forum of Neuroscience Poster Molecular and cellular mechanisms in astrocyte-T cell cross-talk 05.07.2014 Milan, Italy Filipello Fabia;
Annual Meeting Fondazione Italiana Sclerosi Multipla (FISM) Poster Molecular and cellular mechanisms in astrocyte-T cell cross-talk 28.05.2014 Rome, Italy Filipello Fabia;
IX National Conference of the Italian Society of Immunology, Clinical Immunology and Allergology Talk given at a conference Ectonucleotidase activity and Adenosine in the regulation of adaptive immune response 28.05.2014 Florence, Italy Faliti Caterina Elisa;
IX National Conference of the Italian Society of Immunology, Clinical Immunology and Allergology Poster Purinergic P2X7 receptor activity in the regulation of glucose metabolism at the interface between gut-associated lymphoid tissue and commensal microflora 28.05.2014 Florence, Italy Perruzza Lisa;
Italian-German Purine Club Meeting Talk given at a conference Extracellular nucleotides in astrocyte-T cell interaction 20.09.2013 Rimini, Italy Filipello Fabia;
2nd International Aegean Conference on ImmunoMetabolism: Molecular and Cellular Immunology of Metabolism Talk given at a conference Regulation of mucosal IgA response and host-microbiota mutualism in the small intestine by endoluminal ATP 15.09.2013 Rhodes, Greece Grassi Fabio;
15th International Congress of Immunology 2013 Poster Ectonucleotidase activity in CSR of murine B cells 22.08.2013 Milan, Italy Faliti Caterina Elisa;
15th International Congress of Immunology 2013 Poster ATP- mediated Interaction Between Astrocytes and T Cells 22.08.2013 Milan, Italy Filipello Fabia;
15th International Congress of Immunology 2013 Poster TRPM7 kinase activity plays an essential role in T cell colonization of small intestine epithelium 22.08.2013 Milan, Italy Romagnani Andrea;
15th International Congress of Immunology 2013 Poster Complexity of purinergic P2X7 receptor activity in the pathophysiology of Type 1 Diabetes 22.08.2013 Milan, Italy Perruzza Lisa;
13th Italian Neuroimmunology Symposium Talk given at a conference Purinergic P2X signaling in T cell physiology 31.01.2013 Sinalunga (SI), Italy Grassi Fabio;
European Academy of Dermatology and Venereology 4th Scientific Meeting “From the benchside to the clinic and beyond” Talk given at a conference Regulation of Ig secretory response in the small intestine by endoluminal ATP 08.11.2012 Bellinzona, Switzerland Grassi Fabio;
Italian Society of Immunology Clinical Immunology and Allergology (SIICA) Poster ATP–mediated interaction between astrocytes and T cell 05.11.2012 Rozzano, Italy Filipello Fabia;
Congresso Società Italiana Neuroimmunologia (AINI) Poster ATP–mediated interaction between astrocytes and T cell 26.09.2012 Catania, Italy Filipello Fabia;
Purine Club Annual Meeting Talk given at a conference ATP–mediated interaction between astrocytes and T cell 13.09.2012 Pisa, Italy Filipello Fabia;
Convegno Associazione Giovani con Diabete Talk given at a conference Tissue specific immunosuppression by regulatory T cells in type 1 diabetes 26.05.2012 Catania, Italy Grassi Fabio;


Communication with the public

Communication Title Media Place Year
New media (web, blogs, podcasts, news feeds etc.) An IRB team has discovered a regulatory mechanism for gut associated immune system International 2014

Associated projects

Number Title Start Funding scheme
159491 Systemic impact of purinergic P2X7 receptor activity at the interface between gut-associated lymphoid tissue and commensal microflora 01.04.2015 Project funding
145038 Acquisition of a 2-Photon microscope for intravital analysis 01.12.2012 R'EQUIP
124745 Purinergic signaling in T cell physiology 01.04.2009 Project funding

Abstract

In physiological conditions distinct CD4+ T cell subsets coordinate the adaptive immune response against pathogens without causing harm to the organism. In CD4+ T cells adenosine triphosphate (ATP) released upon T cell receptor (TCR) stimulation contributes to mitogen activated protein kinase (MAPK) activation through purinergic P2X receptors. Immunosuppression through CD4+ regulatory T cells (Tregs) expressing the transcription factor Foxp3 constitutes a crucial mechanism to avoid autoimmune reactions toward self-tissue components. High expression of P2X7 is part of the transcriptional signature of Tregs and P2X7 activation by ATP inhibits Tregs suppressive potential. P2X7 mediated signalling in inflammatory microenvironments determine Tregs lineage instability and conversion to pro-inflammatory IL-17 secreting TH17 effector cells. Conversely, blockade of P2X signalling in naïve T cells promotes conversion into immunosuppressive Tregs. Thus, ATP acts as an autocrine factor that integrates stimuli from the microenvironment and cellular energetics through P2X stimulation to tune adaptive immune responses. The first two aims of the present project will be devoted to the drawing of a comprehensive picture of P2X7 signaling in CD4+ T cells. Since P2X7 stimulation was shown to activate phospholipase D (PLD) in various cell types, including T cells, we will analyse PLD activity in activated wild-type (WT) and p2rx7-/- T cells in the presence of P2X7 agonists. Erk phosphorylation and mammalian target of rapamycin (mTOR) activation as possible effectors of P2X7-mediated PLD activation will be investigated. The spatial distribution of signaling molecules within the cell has emerged as a critical factor in determining downstream outcome/s. In Jurkat cells TCR stimulation induces N-ras activation at the Golgi apparatus. Whether Ras-GTP subcellular routing is influenced by purinergic signaling has not been investigated so far. To address whether P2X7 signaling might affect this aspect of Ras signaling we will study the spatial distribution (i.e. plasma membrane vs Golgi recruitment) of Ras in WT and p2rx7-/- T cells. Finally, as a quantitative proteomic approach to identify targets of P2X7 signaling in T cells we will use stable isotope labeling by amino acids in cell culture (SILAC) with sorted CD4 naïve cells from WT and p2rx7-/- mice. These cells will be stimulated in vitro with CD3 and CD28 mabs in the presence of P2X7 agonists. For quantitative analysis of P2X7-regulated phosphorylation, we will label cells with one of two different isotope combinations of lysine and arginine. We will enrich phosphopetides from total cellular extracts by immobilized metal ion-affinity chromatography (IMAC) and HPLC-separated peptides will be analysed in mass spectroscopy. This experimental approach will allow compiling a list of P2X7-regulated phosphorylation events, which might be relevant in regulating T cell differentiation and functional skewing.The correct balance between distinct functional subsets of effector and regulatory CD4+ T cells in the gut mucosa is of fundamental importance to orchestrate protective and tolerogenic responses to microorganisms in order to maintain mucosal barrier integrity. Crohn’s disease and ulcerative colitis are examples of chronic inflammatory diseases of the gastrointestinal tract in which a dysregulated immune response towards intestinal bacteria leads to severe inflammatory tissue destruction. The third aim of the project stemmed from the observation of Peyer’s pathces (PP) hypercellularity in p2rx7-/- mice. In particular, we could detect the selective and highly significant increase of follicular B helper T (TFH) cells. This increase correlated with increase in germinal centre (GC) reaction in PP and increase in fecal immunoglobulins A. We will study the role of ATP/P2X7 signaling axis in homeostatic control of TFH cells, germinal centre reaction and more generally adaptive immunity in the small intestine mucosa. To this end we will perform a metagenomic analysis of small intestine from WT and p2rx7-/- mice to establish whether modulation of germinal centre reaction by P2X7 in PP affects the microbial composition. Finally, since we have previously shown that opening of pannexin-1 hemichannels in the plasma membrane allows ATP release in activated T cells, in the fourth aim of the project we will exploit pannexin-1 knock-out mice to address the role of pannexin-1 in the generation of adaptive Tregs both in vitro and in the gut mucosa.
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