Comparative Analysis of Thymic and Blood Treg in Myasthenia Gravis: Thymic Epithelial Cells Contribute to Thymic Immunoregulatory Defects.

The thymus is involved in autoimmune Myasthenia gravis (MG) associated with anti-acetylcholine (AChR) antibodies. In MG, thymic regulatory T cells (Treg) are not efficiently suppressive, and conventional T cells (Tconv) are resistant to suppression. To better understand the specific role of the thymus in MG, we compared the phenotype and function of peripheral and thymic Treg and Tconv from controls and MG patients. Suppression assays with thymic or peripheral CD4 + T cells showed that the functional impairment in MG was more pronounced in the thymus than in the periphery. Phenotypic analysis of Treg showed a significant reduction of resting and effector Treg in the thymus but not in the periphery of MG patients. CD31, a marker lost with excessive immunoreactivity, was significantly reduced in thymic but not blood resting Treg. These results suggest that an altered thymic environment may explain Treg differences between MG patients and controls. Since thymic epithelial cells (TECs) play a major role in the generation of Treg, we co-cultured healthy thymic CD4 + T cells with control or MG TECs and tested their suppressive function. Co-culture with MG TECs consistently hampers regulatory activity, as compared with control TECs, suggesting that MG TECs contribute to the immune regulation defects of MG CD4 + T cells. MG TECs produced significantly higher thymic stromal lymphopoietin (TSLP) than control TECs, and a neutralizing anti-TSLP antibody partially restored the suppressive capacity of Treg derived from co-cultures with MG TECs, suggesting that TSLP contributed to the defect of thymic Treg in MG patients. Finally, a co-culture of MG CD4 + T cells with control TECs restored numbers and function of MG Treg, demonstrating that a favorable environment could correct the immune regulation defects of T cells in MG. Altogether, our data suggest that the severe defect of thymic Treg is at least partially due to MG TECs that overproduce TSLP. The Treg defects could be corrected by replacing dysfunctional TECs by healthy TECs. These findings highlight the role of the tissue environment on the immune regulation.

Cultured Human Thymic-Derived Cells Display Medullary Thymic Epithelial Cell Phenotype and Functionality.

Thymic epithelial cells are one of the main components of the thymic microenvironment required for T-cell development. In this work, we describe an efficient method free of enzymatic and Facs-sorted methods to culture human medullary thymic epithelial cells without affecting the cell phenotypic, physiologic and functional features. Human medulla thymic epithelial cells (mTECs) are obtained by culturing thymic biopsies explants. After 7 days of primo-culture, mTECs keep their ability to express key molecules involved in immune tolerance processes such as autoimmune regulator, tissue-specific antigens, chemokines, and cytokines. In addition, the cells sensor their cultured environment and consequently adjust their gene expression network. Therefore, we describe and provide a human mTEC model that may be used to test the effect of various molecules on thymic epithelial cell homeostasis and physiology. This method should allow the investigations of the specificities and the knowledge of human mTECs in normal or pathological conditions and therefore discontinue the extrapolations done on the murine models.

Both Treg cells and Tconv cells are defective in the Myasthenia gravis thymus: roles of IL-17 and TNF-α.

Myasthenia gravis (MG) is an autoimmune disease in which the thymus frequently presents follicular hyperplasia and signs of inflammation and T cells display a defect in suppressive regulation. Defects in a suppressive assay can indicate either the defective function of Treg cells or the resistance of Tconv cells to suppression by Treg cells. The aim of this study was to determine which cells were responsible for this defect and to address the mechanisms involved. We first performed cross-experiment studies using purified thymic Treg cells and Tconv cells from controls (CTRL) and MG patients. We confirmed that MG Treg cells were defective in suppressing CTRL Tconv proliferation, and we demonstrated for the first time that MG Tconv cells were resistant to Treg cell suppression. The activation of MG Tconv cells triggered a lower upregulation of FoxP3 and a higher upregulation of CD4 and CD25 than CTRL cells. To investigate the factors that could explain these differences, we analyzed the transcriptomes of purified thymic Treg and Tconv cells from MG patients in comparison to CTRL cells. Many of the pathways revealed by this analysis are involved in other autoimmune diseases, and T cells from MG patients exhibit a Th1/Th17/Tfh signature. An increase in IL-17-related genes was only observed in Treg cells, while increases in IFN-γ, IL-21, and TNF-α were observed in both Treg and Tconv cells. These results were confirmed by PCR studies. In addition, the role of TNF-α in the defect in Tconv cells from MG patients was further confirmed by functional studies. Altogether, our results indicate that the immunoregulatory defects observed in MG patients are caused by both Treg cell and Tconv cell impairment and involve several pro-inflammatory cytokines, with TNF-α playing a key role in this process. The chronic inflammation present in the thymus of MG patients could provide an explanation for the escape of thymic T cells from regulation in the MG thymus.

Defects of immunoregulatory mechanisms in myasthenia gravis: role of IL-17.

Deficient immunoregulation is consistently observed in autoimmune diseases. Here, we summarize the abnormalities of the T cell response in autoimmune myasthenia gravis (MG) by focusing on activation markers, inflammatory features, and imbalance between the different T cell subsets, including Th17 and regulatory T cells (T(reg) cells). In the thymus from MG patients, T(reg) cell numbers are normal while their suppressive function is severely defective, and this defect could not be explained by contaminating effector CD127(low) T cells. A transcriptomic analysis of T(reg) cell and conventional T cell (T(conv) ; CD4(+) CD25(-) cells) subsets pointed out an upregulation of Th17-related genes in MG cells. Together with our previous findings of an inflammatory signature in the MG thymus and an overproduction of IL-1 and IL-6 by MG thymic epithelial cells (TEC), these data strongly suggest that T cell functions are profoundly altered in the thymic pathological environment. In this short review we discuss the mechanisms of chronic inflammation linked to the pathophysiology of MG disease.

A noncanonical bromodomain in the AAA ATPase protein Yta7 directs chromosomal positioning and barrier chromatin activity.

Saccharomyces cerevisiae Yta7 is a barrier active protein that modulates transcriptional states at the silent mating locus, HMR. Additionally, Yta7 regulates histone gene transcription and has overlapping functions with known histone chaperones. This study focused on deciphering the functional role of the noncanonical Yta7 bromodomain. By use of genetic and epistasis analyses, the Yta7 bromodomain was shown to be necessary for barrier activity at HMR and to have overlapping functions with histone regulators (Asf1 and Spt16). Canonical bromodomains can bind to acetylated lysines on histones; however, the Yta7 bromodomain showed an association with histones that was independent of posttranslational modification. Further investigation showed that regions of Yta7 other than the bromodomain conferred histone association. Chromatin immunoprecipitation-chip analyses revealed that the Yta7 bromodomain was not solely responsible for histone association but was also necessary for proper chromosomal positioning of Yta7. This work demonstrates that the Yta7 bromodomain engages histones for certain cellular functions like barrier chromatin maintenance and particular Spt16/Asf1 cellular pathways of chromatin regulation.

Saccharomyces cerevisiae Yta7 regulates histone gene expression.

The Saccharomyces cerevisiae Yta7 protein is a component of a nucleosome bound protein complex that maintains distinct transcriptional zones of chromatin. We previously found that one protein copurifying with Yta7 is the yFACT member Spt16. Epistasis analyses revealed a link between Yta7, Spt16, and other previously identified members of the histone regulatory pathway. In concurrence, Yta7 was found to regulate histone gene transcription in a cell-cycle-dependent manner. Association at the histone gene loci appeared to occur through binding of the bromodomain-like region of Yta7 with the N-terminal tail of histone H3. Our work suggests a mechanism in which Yta7 is localized to chromatin to establish regions of transcriptional silencing, and that one facet of this cellular mechanism is to modulate transcription of histone genes.

Pharmacokinetics and metabolism of apigenin in female and male rats after a single oral administration.

The metabolism of apigenin, a weak estrogenic flavonoid phytochemical, was investigated in the rat. After a single oral administration of radiolabeled apigenin, 51.0% of radioactivity was recovered in urine, 12.0% in feces, 1.2% in the blood, 0.4% in the kidneys, 9.4% in the intestine, 1.2% in the liver, and 24.8% in the rest of the body within 10 days. Sex differences appear with regard to the nature of compounds eliminated via the urinary route: immature male and female rats, like mature female rats, excreted a higher percentage of the mono-glucuronoconjugate of apigenin than the mono-sulfoconjugate of apigenin (10.0-31.6% versus 2.0-3.6%, respectively). Mature male rats excreted the same compounds in an inverse ratio (4.9% and 13.9%, respectively). Radioactivity appeared in the blood only 24 h after oral administration. Blood kinetics showed a high elimination half-time (91.8 h), a distribution volume of 259 ml, and a plasmatic clearance of 1.95 ml/h. All of the parameters calculated from these experiments suggested a slow metabolism of apigenin, with a slow absorption and a slow elimination phase. Thus, a possible accumulation of this flavonoid in the body can be hypothesized.

Metabolism of apigenin by rat liver phase I and phase ii enzymes and by isolated perfused rat liver.

The metabolism of apigenin, a low estrogenic flavonoid phytochemical, was investigated in rat using liver models both in vitro (subcellular fractions) and ex vivo (isolated perfused liver). In vitro, phase I metabolism led to the formation of three monohydroxylated derivatives: luteolin which was the major metabolite (K(m) = 22.5 +/- 1.5 microM; V(max) = 5.605 +/- 0.090 nmol/min/mg protein, means +/- S.E.M.), scutellarein, and iso-scutellarein. These oxidative pathways were mediated by cytochrome P450 monooxygenases (P450s). The use of P450 inhibitors and inducers showed that CYP1A1, CYP2B, and CYP2E1 are involved. In vitro studies of phase II metabolism indicated that apigenin underwent conjugation giving three monoglucuronoconjugates and one monosulfoconjugate. Luteolin led to the formation of four monoglucuronoconjugates, two sulfoconjugates, and one methylconjugate identified as diosmetin. Ex vivo during the apigenin perfusion of an isolated rat liver, none of the phase I metabolites could be recovered. In contrast, two monoglucuronoconjugates and one of the sulfoconjugates of apigenin already identified in vitro were recovered. Moreover, two new derivatives were isolated and identified as a diglucuronoconjugate and a glucuronosulfoconjugate. This work provides new data about the metabolism of apigenin and shows the interest value of using various experimental models in metabolic studies.