Autoimmuunregulaator: geeniregulatsiooni ja promootori metülatsiooni iseloomustamine

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Tüümuse medullaarsed epiteelirakud ekspresseerivad AIRE (Autoimmuunne Regulaator) valku, mis suunab perifeersete koespetsiifiliste geenide avatud geeniekspressiooni. Mutatsioonid AIRE geenis toovad endaga kaasa haruldase autoimmuunhaiguse APECED (Autoimmune Polyendocrinopathy Candidiasis Ectodermal Dystrophy), millega kaasneb immunoloogilise tolerantsuse kadumine mitmete endokriinorganite suhtes. Käesolevas töös leidsime, et koespetsiifiliste geenide ekspressioon sõltub Aire geeni koopiate arvust tüümuses ja Aire kui ka koespetsiifilised geenid ekspresseeruvad tüümuse medullas ning neil on sarnane avaldumismuster tüümuse erinevatel arenguetappidel. Aire geeni lisamise tingimustes tõusis koespetsiifiliste geenide mRNA tase. Tüümuse struktuuri lõhkumise tulemusena langes kõigi uuritud geenide ekpressioon. Antud tulemused näitavad, et Aire geenil on otsene roll koespetsiifiliste geenide ekspressioonil ja läbi Aire geeni on võimalik mõjutada tsentraalse tolerantsuse või autoimmuunsuse tekkimist. Kuna tüümuse kemokiinid on olulised tüümusesse sisenevate tümotsüütide migratsioonil, siis uurisime Aire geeni rolli tüümuse kemokiinide tootmises. Leidsime, et tüümuses Aire geeni puudumise tulemusena langevad CCR4 ja CCR7 ligandide tasemed ning CCR4 ligandid on Aire poolt reguleeritud CD80hi mTEC rakkudes. Sarnaselt koespetsiifilistele antigeenidele jälgisid ka tüümuse kemokiinid Aire avaldumismustrit sünnijärgsel perioodil. Aire geeni üleekspresseerimise tingimustes tõusis CCL5, CCL22 and CCL19 kemokiinide tase. Antud leidude puhul on tegemist Aire uudse rolliga tsentraalse tolerantsuse kujunemises. Edasi uurisime AIRE promooteri metülatsiooni ja leidsime, et AIRE promooter oli hüpometüleeritud mTECides ja cTECides ning CpG metülatsiooni tase tõusis AIRE negatiivsetes tümotsüütides. Samas oli AIRE promooter hüpometüleeritud Aire negatiivsetes tüümuse kasvajates ja perifeersetes kudedes. Ning leidsime positiivse korrelatsiooni AIRE ekspressiooni ja AIRE promooteril asuva aktiivse kromatiini märgise H3K4me3 vahel.Thymic medullary epithelial cells express the AIRE (Autoimmune Regulator) gene, which directs the ectopic expression of peripheral tissue-specific antigens (TSAs). Mutations in the AIRE gene cause a rare autoimmune disease APECED (Autoimmune Polyendocrinopathy Candidiasis Ectodermal Dystrophy), accompanied by a loss of immunological tolerance to a number of endocrine organs. We found that TSA expression is dependent on Aire gene copy number in thymus. Aire as well as TSAs were expressed in thymic medulla and had similar expression pattern in the thymus at the different stages of development. TSA expression increased in the conditions of Aire over-expression. The destruction of the thymus structure resulted in a drop of expression of TSAs. These results suggest that the Aire gene has a direct role in TSA expression and via Aire gene it should be possible to modulate the negative selection of autoreactive T cells. Since thymic chemokines are important for the migration of thymocytes into the thymus, we examined the role of the Aire gene on thymic chemokine production. We found that the thymic absence of Aire gene resulted in decreased levels of CCR4 and CCR7 ligands, whereas CCR4 ligands were regulated by Aire in CD80-positive thymic medullary epithelial cells. Similarly to TSAs in the thymus, the chemokines follow the Aire expression pattern after birth. In over-expression of the Aire gene, the expression of CCL5, CCL22 and CCL19 chemokines was also increased. We describe a novel role for Aire in the development of central tolerance. Furthermore, during the studies of genomic methylation signatures in Aire promoter region in different cells and tissues, we found that the AIRE promoter was hypomethylated in thymic epithelial cells but highly methylated in thymocytes. Also, the AIRE promoter was hypomethylated in AIRE-negative thymic epithelial tumors (thymomas) and in several peripheral tissues. Positive correlation was found in human thymic epithelial cells between AIRE expression and an active chromatin mark; the histone H3K4me3 modification at the AIRE promoter

Chagasic Thymic Atrophy Does Not Affect Negative Selection but Results in the Export of Activated CD4+CD8+ T Cells in Severe Forms of Human Disease

Extrathymic CD4+CD8+ double-positive (DP) T cells are increased in some pathophysiological conditions, including infectious diseases. In the murine model of Chagas disease, it has been shown that the protozoan parasite Trypanosoma cruzi is able to target the thymus and induce alterations of the thymic microenvironment and the lymphoid compartment. In the acute phase, this results in a severe atrophy of the organ and early release of DP cells into the periphery. To date, the effect of the changes promoted by the parasite infection on thymic central tolerance has remained elusive. Herein we show that the intrathymic key elements that are necessary to promote the negative selection of thymocytes undergoing maturation during the thymopoiesis remains functional during the acute chagasic thymic atrophy. Intrathymic expression of the autoimmune regulator factor (Aire) and tissue-restricted antigen (TRA) genes is normal. In addition, the expression of the proapoptotic Bim protein in thymocytes was not changed, revealing that the parasite infection-induced thymus atrophy has no effect on these marker genes necessary to promote clonal deletion of T cells. In a chicken egg ovalbumin (OVA)-specific T-cell receptor (TCR) transgenic system, the administration of OVA peptide into infected mice with thymic atrophy promoted OVA-specific thymocyte apoptosis, further indicating normal negative selection process during the infection. Yet, although the intrathymic checkpoints necessary for thymic negative selection are present in the acute phase of Chagas disease, we found that the DP cells released into the periphery acquire an activated phenotype similar to what is described for activated effector or memory single-positive T cells. Most interestingly, we also demonstrate that increased percentages of peripheral blood subset of DP cells exhibiting an activated HLA-DR+ phenotype are associated with severe cardiac forms of human chronic Chagas disease. These cells may contribute to the immunopathological events seen in the Chagas disease

Thymic atrophy in BALB/c acutely infected with <i>T. cruzi</i>.

<p>The histological profiles show the <b>(upper left)</b> normal thymic architecture (C, cortex; M, medulla) and <b>(upper right)</b> marked cortical and medullary atrophy in the thymus of an acutely-infected mouse. <b>(lower left)</b> The numerous metallophilic macrophages are present in the cortico-medullary zone (arrows) of normal thymus. However, following <i>T. cruzi</i> acute infection <b>(lower right)</b> the number and distribution of metallophilic macrophages are changed with the cells dispersed throughout not only the cortico-medullar area but also the cortical region. Infected mice were evaluated herein at day 15 post-infection. These data are representative of two independent experiments using four mice per group.</p

CD4⁺CD8⁺ T cell numbers in thymus of <i>T. cruzi</i> infected OVA TCR transgenic mice.

<p>OVA-TCR transgenic mice noninfected or infected with <i>T. cruzi</i> (9 days post-infection) were injected <i>i.v.</i> with 0.5 ml of 450 mM solution of OVAp323–339 daily for 3 days and thymocytes were isolated on the fourth day for quantification of double-positive CD4⁺CD8⁺ T cells by flow cytometry. For control, mice were injected with 0.5 ml of PBS instead of the cognate antigen as indicated in the histogram. All numbers are given in 10⁶. Each value represents the mean of total DP cell numbers in 4-8 mice per group. Data are expressed as mean ± standard error of triplicate measurements of one out of two representative experiments. *Differences between groups are significant (<i>P</i><0.05). These data are representative of two independent experiments using five mice per group.</p

Activation markers in lymphocytes from thymus and subcutaneous lymph nodes of <i>T. cruzi</i> infected mice.

<p>Mice were infected with <i>T. cruzi</i> and 15 days later lymphocytes were isolated from thymus and subcutaneous lymph nodes. <b>(a)</b> Thymocytes were stained with CD4-PE, CD8-APC and marker-specific fluorescein isothiocyanate-labeled antibodies prior to flow cytometry analysis. The histograms represent the expression of the CD44, CD69 and CD62L markers in total thymocytes (upper panel) and CD4⁺CD8⁺ T cells (lower panel) from chagasic and normal mice. <b>(b)</b> Comparison of the CD62L expression level from normal or chagasic CD4⁺CD8⁺ T cells <i>versus</i> normal single-positive CD4⁺ and CD8⁺ T cells undergoing intrathymic maturation. Panel <b>(c)</b> depicts representative histograms of CD4⁺CD8⁺ from <i>T. cruzi</i> infected subcutaneous lymph nodes (solid lines), compared with naïve CD4⁺ and CD8⁺ T cells obtained from non-infected mice. <b>(d)</b> Representative histograms of CD44 and CD69 expression in CD4⁺CD8⁺ T cells, activated single-positive CD4⁺ and CD8⁺ T cells from subcutaneous lymph nodes obtained from <i>T. cruzi</i> infected mice. Animals were sacrificed on day 15 post-infection. The data are representative of three independent experiments. The values on the upper right corner indicate the mean fluorescent intensity of the expression of the markers from each histogram. Differences between chagasic DP and activated T cells <i>versus</i> naïve T cells are significant (<i>P</i><0.05). These data are representative of three independent experiments using five mice per group.</p

Intrathymic expression of Bim in the course of acute <i>T. cruzi</i> infection.

<p><b>(a)</b> Thymuses were collected from normal or <i>T cruzi</i> infected BALB/c mice (day 15 post-infection). All numbers are given in 10⁶, and each value represents the mean of total thymocyte numbers in 4-8 mice per group. <b>(b)</b> From individual groups of the same experiments, DP cells isolated by cell sorting from thymus of infected mice at the indicated time-points or normal mice as controls were lysed and proteins were solubilized by detergent lysis and immunoprecipitated with anti-Bim antibody for Western blotting analysis probing with the same antibody. The upper blot shows kinetics of the Bim expression from day 10 to 16 post-infection, during the thymic atrophy, whereas the lower panel reveals a blot was probed with actin, applied as a loading control. <b>(c)</b> Optical densitometry of the western blots using NIH Image software, where Bim expression was normalized with the actin expression. The data are representative of three independent experiments. *Differences between control <i>versus</i> infected mice are significant (<i>P</i><0.05). These data are representative of two independent experiments using five mice per group.</p

Intrathymic tissue-restricted antigen expression levels in atrophic thymus during <i>T cruzi</i> infection.

<p><b>(a)</b> Thymuses were collected from normal or <i>T. cruzi</i> infected BALB/c (day 15 post-infection) and expression levels of Aire and TRA genes were analyzed by real-time PCR. The expression of Aire as well as TRA genes was comparable in infected and uninfected thymus. Data are mean ± standard error. of triplicate measurements in one of two representative experiments using five mice per group. <b>(b)</b> Thymuses were stained with anti-Aire antibody and analyzed by immunofluorescence. The histological profiles show the presence of Aire-positive cells (arrows) in <b>(upper left)</b> normal thymus and <b>(upper right)</b> atrophic thymus at day 15 post-infection. The respective staining controls without the primary antibodies are represented in <b>(lower left)</b> and <b>(lower right)</b>. Inserts represent higher magnifications of Aire-positive cells (brown).</p

Modulation of Aire regulates the expression of tissue-restricted antigens

Intrathymic expression of tissue-restricted antigens (TRAs) has been viewed as the key element in the induction of central tolerance and recently, a central role for the autoimmune regulator (Aire) has been suggested in this process. The aim of this study was to establish whether down or up-regulation of Aire leads to alterations in TRA expression and whether this is limited to thymic epithelial cells. This study also characterized whether TRAs follow Aire expression during normal development, and whether thymic microenvironment plays a role in the expression of Aire and TRAs. We did several in vivo and in vitro experiments to manipulate Aire expression and measured expression of four TRAs (Trefoil factor-3, Insulin-2, Major urinary protein-1 and Salivary protein-1) by real-time RT-PCR. Aire had an allele dose-dependent effect on TRA expression in the thymuses of mice from two strains, C57BL/6J and Balb/c, but had no effect on TRA expression in the lymph nodes. In the thymus, Aire and TRAs were both localized in the medulla and were co-expressed during normal development and involution. In the primary stromal cells as well as thymic epithelial cell line, the adenoviral over-expression of Aire resulted in an increase in TRA expression. By manipulating in vitro organ-cultures we showed that thymic microenvironment plays a dominant role in Aire expression whereas TRAs follow the same pattern. The data underline a direct role for Aire in TRA expression and suggest that modulation of Aire has a potential to control central tolerance and autoimmunity