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The function of the AIRE gene and of Micro RNAs in the control of adhesion between medullary thymic epithelial cells with thymocytes

Grant number: 13/17481-1
Support type:Regular Research Grants
Duration: April 01, 2014 - March 31, 2016
Field of knowledge:Biological Sciences - Genetics
Principal Investigator:Geraldo Aleixo da Silva Passos Júnior
Grantee:Geraldo Aleixo da Silva Passos Júnior
Home Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
Assoc. researchers:Silvana Giuliatti

Abstract

The thymus plays a role in the induction of immunological tolerance to tissue related antigens (TRA). Within the thymic medulla, the medullary thymic epithelial cells (mTECs) express hundreds of these TRAs, which, in turn, represent virtually all organs and tissues of the body. Due to the diversity of representation, this phenomenon was called promiscuous gene expression (PGE) which is largely controlled by the Autoimmune regulator (Aire) gene. The negative selection of self-reactive thymocytes is essential for immune tolerance. Thymocytes derived from bone marrow migrate to the thymus and into the medulla of this organ they interact with mTEC cells expressing TRAs (adhesion mTECs- thymocytes). Clones of thymocytes that strongly recognize TRAs presented via MHC-II are then eliminated by apoptosis (negative selection). As the Aire gene controls part of the PGE / TRAs, it is assumed that the action of this gene might influence adhesion mTECs-thymocytes and indirectly apoptosis of thymocytes (negative selection). As this point is still elusive, we raised the following hypotheses for this project: 1) Expression of the Aire gene influences adhesion mTECs-thymocytes and consequently apoptosis of thymocytes, 2) The microRNAs (miRNAs) control adhesion mTECs-thymocytes 3) miRNAs control the expression of TRAs, 4) miRNAs control the expression of the Aire gene. Our broad objective is to study the transcriptional control exerted by Aire on TRAs in mTECs and also the post-transcriptional control exerted by miRNAs and their consequences on adhesion mTECs-thymocytes. We recently demonstrated that murine (M. musculus) cell line mTEC 3.10 (MHCII +, CD80 +) express in culture the Aire gene and that this gene controls a large set of TRAs and many miRNAs. Furthermore, this cell line is able to reproduce in vitro the adhesion mTECs-thymocytes. This consolidates a model system to test these hypotheses: the mTECs-thymocytes adhesion assay. We will use in vitro silencing of Dicer ribonuclease and Aire by siRNA (transient silencing) and also the selection knockout (KO) cells by transfection with the vector CompoZr to knockout Aire and Dicer (permanent gene disruption). Using manipulated mTEC 3.10 cells (siRNA / Aire KO and / or Dicer KO) we will test the participation of Aire and / or miRNAs by the in vitro adhesion assay. The transcriptome (mRNAs) and miRnome (miRNAs) of these cells will be assessed using microarrays in an attempt to identify TRA genes and / or cell adhesion molecules involved. The sequencing (next-generation sequencing) will be used to evaluate the expression of Aire isoforms and also of the TRA 3'UTR regions that could hybridize (or not) with miRNAs. Finally, to confirm the role of miRNAs on Aire, we will use the luciferase reporter assay to test the hybridization of predicted miRNAs to 3'UTR sequence of this gene. The results of this project will contribute to a better understanding of the transcriptional and post-transcriptional control of mTECs-thymocytes adhesion which is an essential process for negative selection and central tolerance induction. (AU)

Articles published in Agência FAPESP about the research grant
Study advances understanding of how autoimmune diseases develop  

Scientific publications (7)
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
MENDES-DA-CRUZ, DANIELLA A.; LEMOS, JULIA P.; PASSOS, GERALDO A.; SAVINO, WILSON. Abnormal T-Cell Development in the Thymus of Non-obese Diabetic Mice: Possible Relationship With the Pathogenesis of Type 1 Autoimmune Diabetes. FRONTIERS IN ENDOCRINOLOGY, v. 9, JUL 12 2018. Web of Science Citations: 0.
SPECK-HERNANDEZ, CESAR A.; ASSIS, AMANDA F.; FELICIO, RAFAELA F.; COTRIM-SOUSA, LARISSA; PEZZI, NICOLE; LOPES, GABRIEL S.; BOMBONATO-PRADO, KARINA F.; GIULIATTI, SILVANA; PASSOS, GERALDO A. Aire Disruption Influences the Medullary Thymic Epithelial Cell Transcriptome and Interaction With Thymocytes. FRONTIERS IN IMMUNOLOGY, v. 9, MAY 7 2018. Web of Science Citations: 0.
PASSOS, GERALDO A.; SPECK-HERNANDEZ, CESAR A.; ASSIS, AMANDA F.; MENDES-DA-CRUZ, DANIELLA A. Update on Aire and thymic negative selection. IMMUNOLOGY, v. 153, n. 1, p. 10-20, JAN 2018. Web of Science Citations: 4.
OLIVEIRA, ERNNA H.; MACEDO, CLAUDIA; COLLARES, CRISTHIANNA V.; FREITAS, ANA CAROLINA; DONATE, PAULA BARBIM; SAKAMOTO-HOJO, ELZA T.; DONADI, EDUARDO A.; PASSOS, GERALDO A. Aire Downregulation Is Associated with Changes in the Posttranscriptional Control of Peripheral Tissue Antigens in Medullary Thymic Epithelial Cells. FRONTIERS IN IMMUNOLOGY, v. 7, NOV 23 2016. Web of Science Citations: 4.
PEZZI, NICOLE; ASSIS, AMANDA FREIRE; COTRIM-SOUSA, LARISSA COTRIM; LOPES, GABRIEL SARTI; MOSELLA, MARITZA SALAS; LIMA, DJALMA SOUSA; BOMBONATO-PRADO, KARINA F.; PASSOS, GERALDO ALEIXO. Aire knockdown in medullary thymic epithelial cells affects Aire protein, deregulates cell adhesion genes and decreases thymocyte interaction. Molecular Immunology, v. 77, p. 157-173, SEP 2016. Web of Science Citations: 5.
PASSOS, GERALDO ALEIXO; MENDES-DA-CRUZ, DANIELLE AREAS; OLIVEIRA, ERNNA HERIDA. The thymic orchestration involving Aire, miRNAs, and cell-cell interactions during the induction of central tolerance. FRONTIERS IN IMMUNOLOGY, v. 6, JUL 14 2015. Web of Science Citations: 13.
MACEDO, CLAUDIA; OLIVEIRA, ERNNA H.; ALMEIDA, RENATA S.; DONATE, PAULA B.; FORNARI, THAIS A.; PEZZI, NICOLE; SAKAMOTO-HOJO, ELZA T.; DONADI, EDUARDO A.; PASSOS, GERALDO A. Aire-dependent peripheral tissue antigen mRNAs in mTEC cells feature networking refractoriness to microRNA interaction. Immunobiology, v. 220, n. 1, p. 93-102, JAN 2015. Web of Science Citations: 10.

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