Altered expression of fragile X mental retardation-1 (FMR1) in the thymus in autoimmune myasthenia gravis

Predisposition to autoimmunity and inflammatory disorders is observed in patients with fragile X-associated syndromes. These patients have increased numbers of CGG triplets in the 5’ UTR region of FMR1 (Fragile X Mental Retardation 1) gene, that affects its expression. FMR1 is decreased in the thymus of myasthenia gravis (MG) patients, a prototypical autoimmune disease. We thus analyzed the number of CGG triplets in FMR1 in MG, and explored the regulatory mechanisms affecting thymic FMR1 expression. We measured the number of CGGs using thymic DNA from MG and controls, but no abnormalities in CGGs were found in MG that could explain thymic decrease of FMR1. We next analyzed by RT-PCR the expression of FMR1 and its transcription factors in thymic samples, and in thymic epithelial cell cultures in response to inflammatory stimuli. In control thymuses, FMR1 expression was higher in males than females, and correlated with CTCF (CCCTC-binding factor) expression. In MG thymuses, decreased expression of FMR1 was correlated with both CTCF and MAX (Myc-associated factor X) expression. Changes in FMR1 expression were supported by western blot analyses for FMRP. In addition, we demonstrated that FMR1, CTCF and MAX expression in thymic epithelial cells was also sensitive to inflammatory signals. Our results suggest that FMR1 could play a central role in the thymus and autoimmunity. First, in relation with the higher susceptibility of females to autoimmune diseases. Second, due to the modulation of its expression by inflammatory signals that are known to be altered in MG thymuses.

The absence of the autoimmune regulator gene (AIRE) impairs the three-dimensional structure of medullary thymic epithelial cell spheroids

Besides controlling the expression of peripheral tissue antigens, the autoimmune regulator (AIRE) gene also regulates the expression of adhesion genes in medullary thymic epithelial cells (mTECs), an essential process for mTEC-thymocyte interaction for triggering the negative selection in the thymus. For these processes to occur, it is necessary that the medulla compartment forms an adequate three-dimensional (3D) architecture, preserving the thymic medulla. Previous studies have shown that AIRE knockout (KO) mice have a small and disorganized thymic medulla; however, whether Aire influences the mTEC-mTEC interaction in the maintenance of the 3D structure has been little explored. Considering that AIRE controls cell adhesion genes, we hypothesized that this gene affects 3D mTEC-mTEC interaction. To test this, we constructed an in vitro model system for mTEC spheroid formation, in which cells adhere to each other, establishing a 3D structure. The effect of Aire on mTEC-mTEC adhesion was evaluated by comparing AIRE wild type (AIREWT) versus Aire KO (AIRE-/-) mTECs. Considering the 3D spheroid model evaluated, we reported that the absence of AIRE disorganizes the 3D structure of mTEC spheroids, promotes a differential regulation of mTEC classical surface markers, and modulates genes encoding adhesion and other molecules.

NF-κB: At the Borders of Autoimmunity and Inflammation

The transcription factor NF-κB regulates multiple aspects of innate and adaptive immune functions and serves as a pivotal mediator of inflammatory response. In the first part of this review, we discuss the NF-κB inducers, signaling pathways, and regulators involved in immune homeostasis as well as detail the importance of post-translational regulation by ubiquitination in NF-κB function. We also indicate the stages of central and peripheral tolerance where NF-κB plays a fundamental role. With respect to central tolerance, we detail how NF-κB regulates medullary thymic epithelial cell (mTEC) development, homeostasis, and function. Moreover, we elaborate on its role in the migration of double-positive (DP) thymocytes from the thymic cortex to the medulla. With respect to peripheral tolerance, we outline how NF-κB contributes to the inactivation and destruction of autoreactive T and B lymphocytes as well as the differentiation of CD4+-T cell subsets that are implicated in immune tolerance. In the latter half of the review, we describe the contribution of NF-κB to the pathogenesis of autoimmunity and autoinflammation. The recent discovery of mutations involving components of the pathway has both deepened our understanding of autoimmune disease and informed new therapeutic approaches to treat these illnesses.

Indispensable epigenetic control of thymic epithelial cell development and function by polycomb repressive complex 2

Thymic T cell development and T cell receptor repertoire selection are dependent on essential molecular cues provided by thymic epithelial cells (TEC). TEC development and function are regulated by their epigenetic landscape, in which the repressive H3K27me3 epigenetic marks are catalyzed by polycomb repressive complex 2 (PRC2). Here we show that a TEC-targeted deficiency of PRC2 function results in a hypoplastic thymus with reduced ability to express antigens and select a normal repertoire of T cells. The absence of PRC2 activity reveals a transcriptomically distinct medullary TEC lineage that incompletely off-sets the shortage of canonically-derived medullary TEC whereas cortical TEC numbers remain unchanged. This alternative TEC development is associated with the generation of reduced TCR diversity. Hence, normal PRC2 activity and placement of H3K27me3 marks are required for TEC lineage differentiation and function and, in their absence, the thymus is unable to compensate for the loss of a normal TEC scaffold.

The Early Postnatal Life: A Dynamic Period in Thymic Epithelial Cell Differentiation

The microenvironments formed by cortical (c) and medullary (m) thymic epithelial cells (TECs) play a non-redundant role in the generation of functionally diverse and self-tolerant T cells. The role of TECs during the first weeks of the murine postnatal life is particularly challenging due to the significant augment in T cell production. Here, we critically review recent studies centered on the timely coordination between the expansion and maturation of TECs during this period and their specialized role in T cell development and selection. We further discuss how aging impacts on the pool of TEC progenitors and maintenance of functionally thymic epithelial microenvironments, and the implications of these chances in the capacity of the thymus to sustain regular thymopoiesis throughout life.

Thymic Epithelial Cell Alterations and Defective Thymopoiesis Lead to Central and Peripheral Tolerance Perturbation in MHCII Deficiency

Major Histocompatibility Complex (MHC) class II (MHCII) deficiency (MHCII-D), also known as Bare Lymphocyte Syndrome (BLS), is a rare combined immunodeficiency due to mutations in genes regulating expression of MHCII molecules. MHCII deficiency results in impaired cellular and humoral immune responses, leading to severe infections and autoimmunity. Abnormal cross-talk with developing T cells due to the absence of MHCII expression likely leads to defects in thymic epithelial cells (TEC). However, the contribution of TEC alterations to the pathogenesis of this primary immunodeficiency has not been well characterized to date, in particular in regard to immune dysregulation. To this aim, we have performed an in-depth cellular and molecular characterization of TEC in this disease. We observed an overall perturbation of thymic structure and function in both MHCII−/− mice and patients. Transcriptomic and proteomic profiling of murine TEC revealed several alterations. In particular, we demonstrated that impairment of lymphostromal cross-talk in the thymus of MHCII−/− mice affects mTEC maturation and promiscuous gene expression and causes defects of central tolerance. Furthermore, we observed peripheral tolerance impairment, likely due to defective Treg cell generation and/or function and B cell tolerance breakdown. Overall, our findings reveal disease-specific TEC defects resulting in perturbation of central tolerance and limiting the potential benefits of hematopoietic stem cell transplantation in MHCII deficiency.

Impact of thymic epithelial primary cilia on T cell development

Primary cilia are ubiquitous hair-like organelles, usually projecting from the apical cell surface. They are essential for the organogenesis and homeostasis of various physiological functions, and their dysfunction leads to a plethora of human diseases. However, there are few reports on the role of primary cilia in the immune system; therefore, we focused on their role in the thymus that nurtures immature lymphocytes to full-fledged T cells. After detecting primary cilia on the thymic epithelial cell (TEC) expressed transforming growth factor β (TGF-β) receptor in the basal body, we established a line of an intraflagellar transport protein 88 (Ift88) knockout mice lacking primary cilia in TECs (Ift88 TEC null mutant) to clarify their precise role in thymic organogenesis and T cell differentiation. The Ift88-TEC null mutant mice showed stunted cilia or lack of cilia in TECs. The intercellular contact between T cells and the “thymic synapse” of medullary TECs was slightly disorganized in Ift88-TEC null mutants. Notably, the CD4- and CD8-single positive thymocyte subsets increased significantly. The absence or disorganization of thymic cilia downregulated the TGF-β signaling cascade, increasing the number of single positive thymocytes.