Maria Pilar Armengol

The chemokine network. II. On how polymorphisms and alternative splicing increase the number of molecular species and configure intricate patterns of disease susceptibility.

In this second review on chemokines, we focus on the polymorphisms and alternative splicings and on their consequences in disease. Because chemokines are key mediators in the pathogenesis of inflammatory, autoimmune, vascular and neoplastic disorders, a large number of studies attempting to relate particular polymorphisms of chemokines to given diseases have already been conducted, sometimes with contradictory results. Reviewing the published data, it becomes evident that some chemokine genes that are polymorphic have alleles that are found repeatedly, associated with disease of different aetiologies but sharing some aspects of pathogenesis. Among CXC chemokines, single nucleotide polymorphisms (SNPs) in the CXCL8 and CXCL12 genes stand out, as they have alleles associated with many diseases such as asthma and human immunodeficiency virus (HIV), respectively. Of CC chemokines, the stronger associations occur among alleles from SNPs in CCL2 and CCL5 genes and a number of inflammatory conditions. To understand how chemokines contribute to disease it is also necessary to take into account all the isoforms resulting from differential splicing. The first part of this review deals with polymorphisms and the second with the diversity of molecular species derived from each chemokine gene due to alternative splicing phenomena. The number of molecular species and the level of expression of each of them for every chemokine and for each functionally related group of chemokines reaches a complexity that requires new modelling algorithms akin to those proposed in systems biology approaches.

Thyroglobulin Peptides Associate In Vivo to HLA-DR in Autoimmune Thyroid Glands

Endocrine epithelial cells, targets of the autoimmune response in thyroid and other organ-specific autoimmune diseases, express HLA class II (HLA-II) molecules that are presumably involved in the maintenance and regulation of the in situ autoimmune response. HLA-II molecules thus expressed by thyroid cells have the “compact” conformation and are therefore expected to stably bind autologous peptides. Using a new approach to study in situ T cell responses without the characterization of self-reactive T cells and their specificity, we have identified natural HLA-DR-associated peptides in autoimmune organs that will allow finding peptide-specific T cells in situ. This study reports a first analysis of HLA-DR natural ligands from ex vivo Graves’ disease-affected thyroid tissue. Using mass spectrometry, we identified 162 autologous peptides from HLA-DR-expressing cells, including thyroid follicular cells, with some corresponding to predominant molecules of the thyroid colloid. Most interestingly, eight of the peptides were derived from a major autoantigen, thyroglobulin. In vitro binding identified HLA-DR3 as the allele to which one of these peptides likely associates in vivo. Computer modeling and bioinformatics analysis suggested other HLA-DR alleles for binding of other thyroglobulin peptides. Our data demonstrate that although the HLA-DR-associated peptide pool in autoimmune tissue mostly belongs to abundant ubiquitous proteins, peptides from autoantigens are also associated to HLA-DR in vivo and therefore may well be involved in the maintenance and the regulation of the autoimmune response.

Autoimmune Predisposition in Down Syndrome May Result from a Partial Central Tolerance Failure due to Insufficient Intrathymic Expression of AIRE and Peripheral Antigens

Down syndrome (DS), or trisomy of chromosome 21, is the most common genetic disorder associated with autoimmune diseases. Autoimmune regulator protein (AIRE), a transcription factor located on chromosome 21, plays a crucial role in autoimmunity by regulating promiscuous gene expression (pGE). To investigate if autoimmunity in DS is promoted by the reduction of pGE owing to dysregulation of AIRE, we assessed the expression of AIRE and of several peripheral tissue-restricted Ag genes by quantitative PCR in thymus samples from 19 DS subjects and 21 euploid controls. Strikingly, despite the 21 trisomy, AIRE expression was significantly reduced by 2-fold in DS thymuses compared with controls, which was also confirmed by fluorescent microscopy. Allele-specific quantification of intrathymic AIRE showed that despite its lower expression, the three copies are expressed. More importantly, decreased expression of AIRE was accompanied by a reduction of pGE because expression of tissue-restricted Ags, CHRNA1, GAD1, PLP1, KLK3, SAG, TG, and TSHR, was reduced. Of interest, thyroid dysfunction (10 cases of hypothyroidism and 1 of Graves disease) developed in 11 of 19 (57.9%) of the DS individuals and in none of the 21 controls. The thymuses of these DS individuals contained significantly lower levels of AIRE and thyroglobulin, to which tolerance is typically lost in autoimmune thyroiditis leading to hypothyroidism. Our findings provide strong evidence for the fundamental role of AIRE and pGE, namely, central tolerance, in the predisposition to autoimmunity of DS individuals.

Expression of Transporter Associated With Antigen Processing–1 in the Endocrine Cells of Human Pancreatic Islets: Effect of Cytokines and Evidence of Hyperexpression in IDDM

A possible role of transporter associated with antigen processing (TAP)-l in the pathogenesis of IDDM has been investigated by examining the level of TAP-1 expression in the islets of IDDM pancreas and by studying in vitro the effect of interferon (IFN)-γ, IFN-α, and tumor necrosis factor-α in TAP-1 expression by cultured islet cells. A remarkable hyperexpression of TAP-1 has been found in the endocrine cells (β and non-β) of IDDM islets, which constitutes first evidence of hyperexpression of this molecule in the target organ of an autoimmune disease. TAP-1 hyperexpression correlated clearly with HLA class I hyperexpression but only very partially with HLA class II ectopic expression. IFN-γ and IFN-α, both cytokines putatively implicated in IDDM pathogenesis, were capable of inducing TAP-1 protein (as assessed by immunofluorescence flow cytometry) and message (by Northern blot analysis and reverse transcription polymerase chain reaction). These findings suggest that under the influence of cytokines (most probably IFN-α) β-cells may express in their surface a high density of HLA class I–peptide complexes that may facilitate their recognition and lysis by low-affinity CD8+ T-cells.

Influx of recent thymic emigrants into autoimmune thyroid disease glands in humans

Autoimmune thyroid diseases (AITD) are considered as prototypic organ‐specific autoimmune diseases, yet their underlying aetiology remains poorly understood. Among the various pathophysiological mechanisms considered, a failure of central tolerance has received little attention. Here we present evidence in favour of dysregulated thymic function playing a role in AITD. Flow‐cytometric analyses conducted in peripheral blood lymphocytes from 58 AITD patients and 48 age‐ and‐sex‐matched controls showed that AITD patients have significantly higher blood levels of CD4+CD45RA+, CD4+CD31+ and CD4/CD8 double‐positive T lymphocytes, all markers of recent thymic emigrants (RTE). In addition, the α‐signal joint T cell receptor excision circles (TRECs) content (a molecular marker of RTEs) was higher in the group of AITD patients older than 35 years than in age‐matched controls. This was independent from peripheral T cell expansion as assessed by relative telomere length. Comparisons of TREC levels in peripheral blood lymphocytes and intrathyroidal lymphocytes in paired samples showed higher levels within the thyroid during the initial 30 months of the disease, indicating an influx of RTE into the thyroid during the initial stages of AITD. Additionally, a lack of correlation between TREC levels and forkhead box P3 expression suggests that the intrathyroidal RTE are not natural regulatory T cells. These results uncover a hitherto unknown correlation between altered thymic T cell export, the composition of intrathyroidal T cells and autoimmune pathology.

Immunological senescence and thymic function in transplantation.

In the field of organ transplantation, the state of thymic function has not been a major concern but data from bone marrow transplantation studies have unravel the persistence of some thymopoiesis in the adult and, more importantly, the possibility of reinducing it. Given the central role of the thymus in tolerance, these facts have stimulated the interest in the biology of the thymus in humans. Contemporarily, basic research has provided new tools, if imperfect, to monitor thymic function, that is, T-cell receptor excision circles, markers for lymphocytes recently emigrated from the thymus and new imaging techniques. The deployment of these new tools is already changing some paradigms and has now established that re-enactment of thymic activity in the course of bone marrow transplantation or in patients with human immunodeficiency virus on highly active anti-retroviral therapy is beneficial and that can be achieved in the adult. Clinical trials using thymopoiesis-stimulating factors are underway. On the other hand, the discovery that the thymus contains a broad representation of self-antigens and that this depends on the expression of the product of the gene AIRE by the medullary thymic epithelial cells opens the possibility of manipulating central tolerance. Current protocols inducing microchimerism to generate tolerance to solid organ grafts suggest that this could be a feasible therapeutic goal. Therefore, there are many signs indicating that a period of translational research applying the principles of thymic biology and central tolerance to transplantation has already started.

One-tube-PCR technique for CCL2, CCL3, CCL4 and CCL5 applied to fine needle aspiration biopsies shows different profiles in autoimmune and non-autoimmune thyroid disorders

Autoimmune thyroid diseases are characterized by lymphocytic infiltration of the thyroid gland. Chemokines are crucial in the recruitment of lymphocytes and might play an important role in the pathogenesis of autoimmune thyroid disease. The aim of this study was to test the feasibility of analysing by one-tube reverse-transcriptase polymerase chain reaction (RT-PCR) technique CC chemokine profiles in samples obtained by fine needle aspiration biopsy (FNAB). In 27 out of 35 (77%) samples, the material was sufficient for analysis and in 16 (59%) chemokines were detected, thus demonstrating the potential of this technique. Moreover, even in this small group, a statistically significant increase of CCL3 and CCL4 was found in samples from patients with autoimmune thyroid disease as compared to those with multinodular goiter. Chemokine profile measured by improved multiamplification techniques in FNAB thyroid samples may become a useful complementary tool for the management of thyroid autoimmune disease as it constitutes a source of data for research of their pathogenesis.

Decreased AIRE and promiscuous gene expression in thymus from Down syndrome individuals may explain predisposition to autoimmunity

Down syndrome (DS), a chromosomal condition caused by the trisomy of chromosome 21, is associated with various immunological impairments, including a high incidence of autoimmune diseases (e.g., hypothyroidism, type 1 diabetes) and anatomical changes in the thymus. The autoimmune regulator (AIRE) is a transcription factor whose gene maps in 21q22.3 and controls the ectopic expression of a large set of peripheral tissue antigen genes (PTA) in medullary thymic epithelial cells (mTECs). This phenomenon, termed promiscuous gene expression (pGE), plays a key role in central tolerance as demonstrated by the inactivation of AIRE resulting in the rare recessive autoimmune polyendocrinophathy-candidiasis-ectodermal dystrophy syndrome (APECED). DS patients carry three copies of AIRE which may result in its overexpression or, more rarely, its underexpression. To investigate the possibility that DS associated autoimmunity is favored by the impairment of pGE as a consequence of changes in AIRE expression and/or anatomical disorganisation of the thymus, we investigated AIRE, PTA and cell marker gene expression in thymi of DS individuals vs controls. Gene expression was assessed on cDNA from 19 DS and 21 controls total thymus samples by qPCR using TaqMan probes. Interestingly, AIRE gene expression was significantly reduced in thymus from DS vs controls (p = 0.0003). 7 PTA genes (CHRNA1, GAD1, PLP1, KLK3, SAG, TG and TSHR) were also reduced, more markedly for KLK3 and SAG (P = 0.002 and 0.0004 respectively). This changes seems unlikely to result from a reduction in the number of epithelial thymic cell since the levels of keratin expression were not reduced. Allele-specific quantification of three alleles of the AIRE gene demonstrated that, in spite of its overall reduction, the three copies of AIRE genes are expressed in the thymus of DS patients, thus indicating that reduction of AIRE expression is not caused by allele silencing (imprinting). Conclusion, autoimmunity associated to DS is probably in part caused by a reduction of AIRE expression that results in an impaired pGE rather that by a reduction of thymic epithelial cells as part of anatomical disorganization.

A SNP in intron 1 of TSHR controls its thymic expression and susceptibility to Graves’ disease suggesting central tolerance failure in pathogenesis

Graves’ disease (GD) is the paradigm of an anti-receptor autoimmune disease with agonistic auto-antibodies against the thyrotropin receptor (TSHR) being the underlying pathogenic mechanism. TSHR belongs to the category of tissue-restricted antigens (TRAs), which are expressed by medullary thymic epithelial cells (mTECs) and thereby induce central T cell tolerance. In order to understand the association between TSHR gene polymorphisms and GD we tested the hypothesis that TSHR gene variants affect susceptibility to GD by influencing levels of TSHR transcription in the thymus. The results indicate that thymic glands from normal children homozygous for the rs179247 predisposing allele of TSHR had significantly fewer TSHR mRNA transcripts than carriers of the protective allele. In addition, in heterozygous, the TSHR predisposing allele was expressed at a lower level than the protective one as demonstrated by Allele Specific Transcript Quantification. The effect of TSHR SNP rs179247 was thymus-specific and not observed in thyroid glands. These results constitute first evidence for the involvement of central tolerance in the loss of tolerance to TSHR in GD and underscore the concept that variable expression levels of major target autoantigens in the thymus influence the predisposition to autoimmunity presumably by changing the threshold of tolerance.