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Changes in how a gene functions may impair the selection of lymphocytes in the thymus and cause autoimmune diseases

The garden herb thyme (Thymus vulgaris), whose shape must have inspired Galen to give the name thymus to the gland behind the breastbone

LÉO RAMOSThe garden herb thyme (Thymus vulgaris), whose shape must have inspired Galen to give the name thymus to the gland behind the breastbone LÉO RAMOS

Ernna Oliveira, a biologist, endured six months of failure and frustration before she was able to successfully inject a red molecular solution into the thymus gland of living mice. The solution is able to inhibit the action of genes, but the tricky part was to inject it without having it extend to the lungs or heart, two neighboring organs. “Today she does this so skillfully,” says Geraldo Passos, the coordinator of the laboratory at the University of São Paulo (USP) in Ribeirão Preto where she works. Through this and other experiments, Passos, Oliveira and other USP researchers have expanded our knowledge of the origin of autoimmune diseases, such as Type 1 diabetes, lupus, rheumatoid arthritis and vitiligo, in which immune cells attack the body’s healthy cells instead of destroying only invading microorganisms.

Ten years ago we knew that these diseases were the result of harmful changes—or mutations—in a gene found in the thymus and known as the AIRE autoimmune regulator. When it’s functioning as it should, this gene produces a protein that selects defense cells, preventing them from attacking the body’s normal cells. The USP team found that autoimmune diseases could also result from the irregular activity of this gene, even without mutations, thereby reinforcing the findings of other research groups, mainly in the United States. “It’s not all or nothing,” says Passos. “The AIRE gene can be perfect, but small changes in the way it functions apparently can also lead to autoimmunity.”

The work of the USP Ribeirão Preto team has contributed to a reevaluation of the thymus, which before was seen only as a place for maturation of a type of blood cell, T lymphocytes, but it is now considered an essential organ in the body’s defense system. “Tumors of the thymus or injuries caused by cardiac surgery, since the thymus is situated in front of the heart, can be very harmful to the entire body,” notes Antonio Condino Neto, a researcher at the Institute of Biomedical Sciences at USP (ICB/USP) in São Paulo, who also works in this specialty.

Defects in how the thymus functions may cause or worsen some 30 different diseases, and not just autoimmune diseases. These include leukemia and other cancers, intestinal or heart inflammation, in addition to some forms of primary immunodeficiency, a cluster of about 180 rare and highly lethal diseases, characterized by a malfunctioning of the body’s immune system (see Pesquisa FAPESP Issues nº 191 and nº 206). At the Oswaldo Cruz Foundation (Fiocruz) in Rio de Janeiro, the team led by Wilson Savino found that atrophy of the thymus is a common feature of infectious diseases caused by viruses or bacteria. In addition, and of concern to doctors, common occurrences such as the prolonged use of large doses of corticosteroids and other drugs that inhibit the body’s defense system can also reduce thymic activity; it is not known whether recovery is complete after discontinuation of the treatment.

Attack only the enemies
If the immune cells attack the body itself, most likely something has gone wrong with the AIRE gene, which produces a protein, also called AIRE, that operates intensely in the cell nuclei of the thymus. This protein controls the production of a number of the body’s proteins, such as insulin and collagen. “The thymus is a kind of body protein library,” says Passos. The presentation of these proteins to T lymphocytes, which mature in the thymus, after being produced in bone marrow, teaches them to recognize what is the body’s own and thus should be spared. “The T lymphocytes that attack these proteins are eliminated before migrating into the bloodstream. Those that do not recognize the proteins will pass the test and be released into circulation, because they will be able to attack only microbes and tumors, which are considered foreign elements.”

An integrated network: representation of the lymphatic system (in green), important in the body’s defense, the thymus (in yellow, mid-chest) and the spleen (right)

Roger Harris / Science Photo Library An integrated network: representation of the lymphatic system (in green), important in the body’s defense, the thymus (in yellow, mid-chest) and the spleen (right)Roger Harris / Science Photo Library

It’s not always so. The AIRE gene and its protein may be perfect with no mutations, but disturbances in how it functions may impair the selection of T lymphocytes in the thymus. At USP in Ribeirão Preto, one of the experiments that led to this conclusion was that of Ernna Oliveira, who injected molecules to inhibit gene expression, known as RNA interference (RNAi), into the thymus glands of mice. This resulted in a reduction of up to 60% in AIRE gene activity. The production of other proteins of the thymus and unwanted T lymphocytes, which should have been eliminated, gained access to the bloodstream, migrated to the pancreas and destroyed the cells that produce insulin, resulting in Type 1 diabetes.

In 2009 a team from Harvard University demonstrated that deficiencies in the AIRE gene could produce the same effect, the destruction of the beta cells of the pancreas, and could cause diabetes in a strain of laboratory mouse that naturally develops Type 1 diabetes. That was an indication that this gene is essential to preventing an attack on proteins or cells of the body, the so-called aggressive autoimmunity. Passos believes that the mechanism seen in Type 1 diabetes could also be a cause of other autoimmune diseases such as lupus erythematosus, characterized by lesions on the skin and internal organs, and rheumatoid arthritis, characterized by destruction of the collagen of joints.

In another study, the team led by Passos collaborated with Dr. Eduardo Donadi, a physician, and Elza Sakamoto-Hojo, a biologist, also with USP-Ribeirão Preto. They examined the gene expression of immune cells in the blood of 56 people with diabetes—19 with Type 1 diabetes, 20 with Type 2 characterized by insulin resistance, found primarily in obese patients, and 17 with a form of diabetes developed only during pregnancy. Among their conclusions, they found that diabetes and inflammation are phenomena having a close association; most likely each aggravates the other. Furthermore, the profile of genes activated in Type 1 diabetes supported the hypothesis that the disease’s origin was indeed the failure to eliminate T-lymphocytes that attack the pancreas and destroy the cells that produce insulin.

Against fungi
The protein of the AIRE gene may act not only in cell nuclei of the thymus, inducing the production of other proteins that participate in the selection of T lymphocytes, but also in the cytoplasm of the cells of the thymus, favoring or harming the defense against disease-causing microorganisms, according to the most recent experiments of Condino Neto’s team. This group examined the potential origins of susceptibility to chronic infections caused by the fungus Candida albicans, a characteristic that remained unexplained for a rare hereditary syndrome known as autoimmune polyendocrinopathy candidiasis-ectodermal dystrophy (APECED), caused by a mutation in the AIRE gene.

Proof of concept: RNAi molecules (in red) enter the thymus and alter the expression of the AIRE gene

FMRP-USP Immunogenetics Laboratory Proof of concept: RNAi molecules (in red) enter the thymus and alter the expression of the AIRE geneFMRP-USP Immunogenetics Laboratory

Through experiments using cultured human cells, Luis Alberto Pedrosa, of Condino Neto’s team, concluded that an abnormal version of the protein, resulting from a mutation in the AIRE gene, fails to activate the dectin 1 protein, which is essential to enabling a type of cell found in the thymus, macrophages, which recognize and attack fungi, including Candida albicans. Thus, the AIRE gene protein may not only act as the so-called transcription factor inducing the production of other proteins, but also stimulate another defense mechanism, innate immunity, made up of cells skilled in identifying and destroying disease-causing microorganisms.

This is yet another surprise in the history of the thymus. One of the fathers of Western medicine, Galen of Pergamon, who lived between the years 130 and 210, was the first to describe an organ located just below the breastbone to which he gave the name thymus because it resembled the garden herb thyme (Thymus vulgaris). Because of its proximity to the heart, Galen thought it could be the center of courage and affection. It was not until 1961 that the French physician Jacques Miller demonstrated its role in the maturation and selection of lymphocytes. The origin of autoimmune diseases would become clearer two years later.

Maybe in the thymus, if it could be repaired, also lay the answer to solving many diseases. A French and English team used electrical pulses—a technique known as electroporation, the same one used in Ribeirão Preto—to inject DNA into the thymus glands of mice and correct a severe form of primary immunodeficiency. As a result, the thymus began producing normal lymphocytes. “This process applied to the thymus could represent a simple and effective alternative to gene therapy for T cell immunodeficiency,” concluded the team coordinated by Magali Irla and Catherine Nguyen, of the University of the Mediterranean in Marseille, France, in an article published in the journal PLOS One.

1. Control of the transcriptome in diabetes mellitus (nº 2008/56594-8); Grant mechanism Thematic Project; Principal investigator Geraldo Aleixo da Silva Passos Júnior (Ribeirão Preto School of Medicine – FMRP, University of São Paulo); Investment R$ 1,256,782.94 (FAPESP).
2. AIRE deficiency and susceptibility to infection by Candida albicans: new mechanisms (nº 2010/51653-6); Grant Mechanism Regular Line of Research Project Award; Principal investigator Condino Antonio Neto (ICB-USP); Investment R$ 148,013.70 (FAPESP).

Scientific articles
OLIVEIRA, E. H. et al. Expression profile of peripheral tissue antigen genes in medullary thymic epithelial cells (mTECs) is dependent on mRNA levels of autoimmune regulator (Aire). Immunobiology. v. 218, n. 1, p. 96-104. 2013.
IRLA M. et al. ZAP-70 Restoration in mice by in vivo thymic electroporation. PLoS ONE. v. 3, n. 4, e2059, 2008.
PEDROZA L. A. et al. Autoimmune regulator (Aire) contributes to Dectin-1-induced TNF-α production and complexes with caspase recruitment domain-containing protein 9 (Card9), spleen tyrosine kinase (Syk), and Dectin-1. Journal of Allergy and Clinical Immunology. v. 129, n. 2, p. 464-72. 2012.