Induction Of Apoptosis In Thymocytes Research Articles

Glucocorticoids impair T lymphopoiesis after myocardial infarction.

The thymus, where T lymphocytes develop and mature, is sensitive to insults such as tissue ischemia or injury. The insults can cause thymic atrophy and compromise T-cell development, potentially impairing adaptive immunity. The objective of this study was to investigate whether myocardial infarction (MI) induces thymic injury to impair T lymphopoiesis and to uncover the underlying mechanisms. When compared with sham controls, MI mice at day 7 post-MI exhibited smaller thymus, lower cellularity, as well as less thymocytes at different developmental stages, indicative of T-lymphopoiesis impairment following MI. Accordingly, the spleen of MI mice has less T cells and recent thymic emigrants (RTEs), implying that the thymus of MI mice releases fewer mature thymocytes than sham controls. Interestingly, the secretory function of splenic T cells was not affected by MI. Further experiments showed that the reduction of thymocytes in MI mice was due to increased thymocyte apoptosis. Removal of adrenal glands by adrenalectomy (ADX) prevented MI-induced thymic injury and dysfunction, whereas corticosterone supplementation in ADX + MI mice reinduced thymic injury and dysfunction, indicating that glucocorticoids mediate thymic damage triggered by MI. Eosinophils play essential roles in thymic regeneration postirradiation, and eosinophil-deficient mice exhibit impaired thymic recovery after sublethal irradiation. Interestingly, the thymus was fully regenerated in both wild-type and eosinophil-deficient mice at day 14 post-MI, suggesting that eosinophils are not critical for thymus regeneration post-MI. In conclusion, our study demonstrates that MI-induced glucocorticoids trigger thymocyte apoptosis and impair T lymphopoiesis, resulting in less mature thymocyte release to the spleen.NEW & NOTEWORTHY The thymus is essential for maintaining whole body T-cell output. Thymic injury can adversely affect T lymphopoiesis and T-cell immune response. This study demonstrates that MI induces thymocyte apoptosis and compromises T lymphopoiesis, resulting in fewer releases of mature thymocytes to the spleen. This process is mediated by glucocorticoids secreted by adrenal glands. Therefore, targeting glucocorticoids represents a novel approach to attenuate post-MI thymic injury.

Dietary genistein supplementation alters mRNA expression profile and alternative splicing signature in the thymus of chicks with lipopolysaccharide challenge

Genistein is abundant in the soybean products, which exerts prominent effects on immune function. Little information is available about the effect of dietary genistein on thymic transcriptome, especially when suffering from lipopolysaccharide challenge. In this study, 180 one-day-old male broilers were randomly allocated to 3 groups: nonchallenged chicks given a basal diet (CON), and lipopolysaccharide-challenged chicks fed a basal diet (LPS), or lipopolysaccharide-challenged chicks fed a basal diet supplemented with 40 mg/kg genistein (GEN). Lipopolysaccharide injection induced thymocyte apoptosis and inflammatory reactions in the chicks. The results showed dietary genistein significantly reduced the percentage of CD3+ T lymphocytes by 10.04% and CD4+/CD8+ T lymphocyte ratio by 21.88% in the peripheral blood induced by lipopolysaccharide injection (P < 0.05). In addition, genistein significantly reduced the thymus index by 50% and apoptotic index by 12.34% induced by LPS challenge (P < 0.05). Transcriptomic analysis identified 1,926 DEGs (1,014 upregulated and 912 downregulated, P < 0.05) between GEN and LPS groups, which altered the mRNA expression profile and signaling pathways (Toll-like receptor, and NOD-like receptor signaling pathway) in the thymus. Furthermore, 5 splicing (AS) isoforms of the Drosophila Disabled-2 (DAB2) gene were detected, which were significantly upregulated in the GEN group compared with that in the LPS group. In summary, dietary genistein supplementation altered the RNA expression profile and AS signatures in the thymus, and alleviated immune response against lipopolysaccharide challenge.

Mixed Lineage Kinase 3 Promotes Dexamethasone-Induced Thymocyte Apoptosis

Abstract Dexamethasone (DEX) is a corticosteroid known to regulate T-lymphocyte growth/differentiation and used in the clinic as an anti-inflammatory and immunosuppressant agent. The glucocorticoid hormones, including DEX, can induce thymocyte apoptosis. It has been reported that DEX-induced thymocyte apoptosis is associated with an early stage ceramide generation. Since we reported earlier that ceramide induces Mixed Lineage Kinase 3 (MLK3) activation and therefore, we probed whether MLK3 inhibition could attenuate DEX-induced thymocyte apoptosis. Here, we report that genetic loss of MLK3, a member of MAP3K family attenuates DEX-induced apoptosis in thymocytes. To understand any differences in thymic T cell surface markers at the basal level between WT and MLK3−/− mice, we determined protein expression of various surface proteins involved in T cell activation and apoptosis. Flow cytometry analyses indicated that the genetic loss of MLK3 increases CD27 expression on thymic CD4+, CD8+, and CD4+CD8+ T cells; however, CD28 expression was mainly increased on CD8+ and CD4+CD8+ T cells due to loss of MLK3. Interestingly, genetic loss of MLK3 paradoxically regulated CD70 expression on thymic and splenic T cells. Furthermore, 4–5 weeks old WT and MLK3−/− mice were challenged with DEX for 24 h, and apoptosis in thymic T cells was determined. The flow cytometry results showed a significant decrease in the DEX-induced apoptosis in thymic CD4+, CD8+, and CD4+CD8+ T cells, isolated from MLK3−/− mice. Taken together, these results suggest that MLK3 could be a potential target for DEX-induced thymocyte apoptosis and its inhibitors can be used to mitigate thymocyte apoptosis.

Excessive fluoride exposure induces thymocyte apoptosis and impairs cell division: Roles of the PERK and IRE1 pathways

• Excessive fluoride inhibited division and induced apoptosis of thymocytes. • Excessive fluoride induced activation of PERK and IRE1 signaling in thymocytes. • PERK and IRE1 signaling induced by fluoride plays a key role in immunosuppression.

MicroRNA-653 Inhibits Thymocyte Proliferation and Induces Thymocyte Apoptosis in Mice with Autoimmune Myasthenia Gravis by Downregulating TRIM9

Objectives: Myasthenia gravis (MG) is an organ-specific autoimmune neuromuscular disorder that occurs as a result of the impairment in neuromuscular junction and autoantibody attack on the postsynaptic receptors. Increasing evidence suggests that microRNAs (miRs) might be involved in the development of MG. Therefore, the present study aimed to investigate the regulatory function of miR-653 on MG and its relationship with tripartite motif 9 (TRIM9). Methods: The thymic tissues obtained from MG patients with thymic hyperplasia were prepared for establishing an MG mouse model in BALB/c mice. Afterwards, the miR-653 and TRIM9 expressions were determined in thymic tissues. A dual-luciferase reporter assay was carried out to validate whether miR-653 directly targets TRIM9. Finally, the thymocytes were exposed to mimics or inhibitors of miR-653, or siRNA against TRIM9 with the use of MTT assays and flow cytometry for the verification of the gain or loss function of miR-653 and TRIM9 on viability, cell cycle progression, and apoptosis of thymocytes. Results: There was a decrease in thymocyte miR-653 and an increase in TRIM9 in thymic tissues of MG mice. miR-653 was found to negatively regulate TRIM9. Overexpression of miR-653 or depletion of TRIM9 resulted in the inhibition of cell viability, suppression of cell cycle progression, and induction of apoptosis rate in thymocytes. Conclusion: The findings from the present study provided evidence that miR-653 impairs proliferation and promotes apoptosis of thymocytes of MG mice by suppressing TRIM9, indicating that miR-653 could be used as potential therapeutic target in the treatment of autoimmune MG.

Improvement of glucocorticoid-impaired thymus function by dihydromyricetin via up-regulation of PPARγ-associated fatty acid metabolism.

T lymphocytes produced by the thymus are essential mediators of immunity. Accelerated thymic atrophy appears in the patients with administration of glucocorticoids (GCs) which are commonly-used drugs to treat autoimmune and infectious diseases, leading to dysregulation of immunity with manifestation of progressive diminution of new T cell production. However, there is no ideal method to overcome such side effects of GCs. In the current study, we proposed a composition of dexamethasone (DEX) and dihydromyricetin (DMY) derived from a medicinal plant, which could protect from DEX-induced thymus damage and simultaneously enhance the anti-inflammatory effect of DEX. In the current study, we found that DEX-damaged thymic cellularity and architecture, reduced thymocyte numbers, induced thymocyte apoptosis and dropped CD4+ and CD8+ double positive T cell numbers in thymus which was effectively improved by co-treatment with DMY. Quantification of signal joint TCR delta excision circles (TRECs) and Vβ TCR spectratyping analysis were employed to determine the thymus function with indicated treatments. The results showed that DEX-impaired thymus output and decreased TCR cell diversity which was ameliorated by co-treatment with DMY. iTRAQ 2D LC-MS/MS was applied to analyze the proteomic profiling of thymus of mice treated with or without indicated agents, followed by informatics analysis to identify the correlated signaling pathway. After validated by Western blotting and Real-time PCR, we found that PPARγ-associated fatty acid metabolism was increased in the thymic tissues of the animals treated with DMY plus DEX than the animals treated with DEX alone. The agonist and antagonist of PPARγ were further employed to verify the role of PPARγ in the present study. Furthermore, DMY demonstrated a synergistic effect with co-administration of DEX on suppressing inflammation in vivo. Collectively, DMY relieved thymus function damaged by DEX via regulation of PPARγ-associated fatty acid metabolism. Our findings may provide a new strategy on protection of thymus from damage caused by GCs by using appropriate adjuvant natural agents through up-regulation of PPARγ-associated fatty acid metabolism.

Dibromoacetic Acid Induces Thymocyte Apoptosis by Blocking Cell Cycle Progression, Increasing Intracellular Calcium, and the Fas/FasL Pathway in Vitro.

Dibromoacetic acid (DBAA), a haloacetic acid found in drinking water as a disinfection by-product, can cause many adverse effects, including immunotoxicity. In a previous study, we confirmed that DBAA can induce obvious immunotoxicity in mice but that the underlying mechanisms are not clearly understood. In our current study, we confirmed that DBAA induced cytotoxicity and apoptosis in thymocytes isolated from mice by a range of DBAA concentrations (0, 5, 10, 20, or 40 μM). The data showed that DBAA exposure led to a significant decrease in proliferative responses to T-cell mitogens and obvious inhibition in the production of cytokines interleukin-2 and interleukin-4. We found obvious morphological changes of apoptosis in thymocytes and observed the percentage of apoptotic thymocytes to increase significantly as the DBAA concentration increased. Further investigation showed that DBAA can cause G0/G1 arrest in cell cycle analysis, increase intracellular calcium ([Ca(2+)]i) levels, increase the expression of Fas/FasL proteins, and decrease the expression of Bcl-2 protein. It is concluded that in vitro exposure to DBAA can lead to marked cytotoxicity and apoptosis among thymocytes, and the mechanism involved is strongly related to blocking cell cycle progression, increasing intracellular calcium, and increasing Fas/FasL expressions.

Characterizing the thymic lesions in piglets infected with attenuated strains of highly pathogenic porcine reproductive and respiratory syndrome virus

Piglets infected with the highly pathogenic PRRSV (HP-PRRSV) HuN4 strain develop severe thymus atrophy. However, the attenuated strain HuN4-F112 does not lead to lesions in organs. Here, we have characterized the thymic lesions in piglets infected with attenuated strains of HP-PRRSV HuN4 isolated at different passages in the attenuation process to produce HuN4-F112 from the parent HuN4 strain (HuN4-F5, HuN4-F15, HuN4-F23, HuN4-F30, and HuN4-F112). The thymic effects of infection were evaluated in terms of the thymus/body weight ratio, pathological changes, and thymocytes apoptosis. The ability of HP-PRRSV to induce thymus atrophy was reduced following attenuation after 23 passages; the HuN4-F23, but not HuN4-F30, caused thymus atrophy. The ability of the virus to induce thymocyte apoptosis decreased as it became attenuated. In addition, the viral load in the thymus was reduced as the virus was attenuated. The HuN4-F23 and HuN4-F30 strains might provide insight into the molecular mechanisms of HP-PRRSV pathogenesis. Taken together, our results indicate that the ability of HP-PRRSV to induce thymic atrophy is related to its pathogenicity.

Mechanism of immunoprotective effects of curcumin in DLM-induced thymic apoptosis and altered immune function: an in silico and in vitro study

Curcumin, a main component of Curcuma Longa Linn, is a plant polyphenol used as an immune-enhancer in the Indian system of traditional medicine. However, its underlying mechanism of immune-protection remains unknown. The present study is designed to delineate the role of curcumin in deltamethrin (DLM)-induced thymocyte apoptosis and altered immune functions. In silico studies revealed that curcumin has a strong binding affinity toward CD4 and CD8 receptors. DLM (25 µM) induces thymocytes apoptosis through oxidative stress and caspase-dependent pathways. Various concentrations of curcumin (1, 10 and 50 µg/ml), when added along with DLM, caused a concentration- and time-related amelioration in apoptogenic signaling pathways induced by DLM. Inhibition of DLM-induced reactive oxygen species production, replenishment of glutathione and suppression of caspase activities by curcumin may thus be responsible for the suppression of downstream cascade of events, i.e. apoptosis, phenotypic changes and altered cytokine release. Thus, this study clearly demonstrates that the mechanism of immunoprotection of curcumin in DLM-induced thymic apoptosis includes inhibition of oxidative stress and caspase-dependent pathways underlying apoptosis.

In vivo protective effect of betulinic acid on dexamethasone induced thymocyte apoptosis by reducing oxidative stress.

Dexamethasone (Dex), a synthetic glucocorticoid, is strictly controlled for use due to its serious side effects, including immune suppression. Betulinic acid (BA), an antioxidant prepared from the white birch, exhibits immunomodulation properties. To assess the implications and investigate the mechanisms of BA-elicited immunomodulation, we hypothesized that Dex induced thymocyte apoptosis via oxidative stress could be lessened by BA. Mice were given oral doses of BA (0.25, 0.5, and 1.0mg/kg) daily for 14 days, and induced oxidative stress by giving a single dose of Dex intraperitoneal at the dosage of 25mg/kg body weight 8h after the last administration of BA. Dex administration alone significantly decreased antioxidant enzyme activities, while significantly increased reactive oxygen species (ROS) production, lipid peroxidation, mitochondrial dysfunctions, caspase-3 activation and cellular apoptosis. However, pretreatment with BA dose-dependently mitigated Dex-induced oxidative damage after 14 days of feeding. In addition to ROS scavenging activity in Dex-induced thymocyte, BA administration decreased lipid peroxidation, up-regulated antioxidant enzymes, restored mitochondrial function, increased Bcl-2 expression but reduced Bax expression, inhibited caspase-3 activation, and improved cell survival. These findings reveal a protective capability of BA against Dex-induced cell death by reducing oxidative stress via mitochondrial mediated signal pathway which could be the potential mechanism underlying BA-elicited immunomodulation.

Scavenger Receptor BI and High-Density Lipoprotein Regulate Thymocyte Apoptosis in Sepsis

Thymocyte apoptosis is a major event in sepsis; however, how this process is regulated remains poorly understood. Septic stress induces glucocorticoids production which triggers thymocyte apoptosis. Here, we used scavenger receptor BI (SR-BI)-null mice, which are completely deficient in inducible glucocorticoids in sepsis, to investigate the regulation of thymocyte apoptosis in sepsis. Cecal ligation and puncture induced profound thymocyte apoptosis in SR-BI(+/+) mice, but no thymocyte apoptosis in SR-BI(-/-) mice because of lack of inducible glucocorticoids. Unexpectedly, supplementation of glucocorticoids only partly restored thymocyte apoptosis in SR-BI(-/-) mice. We demonstrated that high-density lipoprotein (HDL) is a critical modulator for thymocyte apoptosis. SR-BI(+/+) HDL significantly enhanced glucocorticoid-induced thymocyte apoptosis, but SR-BI(-/-) HDL had no such activity. Further study revealed that SR-BI(+/+) HDL modulates glucocorticoid-induced thymocyte apoptosis via promoting glucocorticoid receptor translocation, but SR-BI(-/-) HDL loses such regulatory activity. To understand why SR-BI(-/-) HDL loses its regulatory activity, we analyzed HDL cholesterol contents. There was 3-fold enrichment of unesterified cholesterol in SR-BI(-/-) HDL compared with SR-BI(+/+) HDL. Normalization of unesterified cholesterol in SR-BI(-/-) HDL by probucol administration or lecithin cholesteryl acyltransferase expression restored glucocorticoid-induced thymocyte apoptosis, and incorporating unesterified cholesterol into SR-BI(+/+) HDL rendered SR-BI(+/+) HDL dysfunctional. Using lckCre-GR(fl/fl) mice in which thymocytes lack cecal ligation and puncture-induced thymocyte apoptosis, we showed that lckCre-GR(fl/fl) mice were significantly more susceptible to cecal ligation and puncture-induced septic death than GR(fl/fl) control mice, suggesting that glucocorticoid-induced thymocyte apoptosis is required for protection against sepsis. The findings in this study reveal a novel regulatory mechanism of thymocyte apoptosis in sepsis by SR-BI and HDL.

Epidermal growth factor-like domain 8 inhibits the survival and proliferation of mouse thymocytes

Thymic epithelial cells (TECs) play a critical role in T-cell development through their intercellular interactions and by producing various soluble proteins, such as growth factors, cytokines and chemokines. In this study, we report a new role for epidermal growth factor-like domain 8 (EGFL8) in the regulation of the survival and proliferation of mouse thymocytes. Mouse recombinant EGFL8 (rEGFL8) protein was produced using an E. coli system and its biological role in mouse thymocytes was determined. The injection of rEGFL8 in mice in vivo resulted in a decrease in the weight of the thymus, as well as in the number of total thymocytes; rEGFL8 also inhibited thymocyte proliferation and induced thymocyte apoptosis. Furthermore, rEGFL8 suppressed the expression of the Notch downstream targets, Hes1 and Hey1, in mouse thymocytes and TECs, indicating that EGFL8 negatively regulates the Notch signaling pathway in these cells. The identification of the role of EGFL8 in thymocytes may aid in the determination of the fate of thymocytes during T-cell development.

Arachidonic acid accumulates in the stromal macrophages during thymus involution in diabetes

Diabetes is a debilitating disease with chronic evolution that affects many tissues and organs over its course. Thymus is an organ that is affected early after the onset of diabetes, gradually involuting until it loses most of its thymocyte populations. We show evidence of accumulating free fatty acids with generation of eicosanoids in the diabetic thymus and we present a possible mechanism for the involution of the organ during the disease. Young rats were injected with streptozotocin and their thymuses examined for cell death by flow cytometry and TUNEL reaction. Accumulation of lipids in the diabetic thymus was investigated by histology and electron microscopy. The identity and quantitation of accumulating lipids was done with gas chromatography-mass spectrometry and liquid chromatography. The expression and dynamics of the enzymes were monitored via immunohistochemistry. Diabetes causes thymus involution by elevating the thymocyte apoptosis. Exposure of thymocytes to elevated concentration of glucose causes apoptosis. After the onset of diabetes, there is a gradual accumulation of free fatty acids in the stromal macrophages including arachidonic acid, the substrate for eicosanoids. The eicosanoids do not cause thymocyte apoptosis but administration of a cyclooxygenase inhibitor reduces the staining for ED1, a macrophage marker whose intensity correlates with phagocytic activity. Diabetes causes thymus involution that is accompanied by accumulation of free fatty acids in the thymic macrophages. Excess glucose is able to induce thymocyte apoptosis but eicosanoids are involved in the chemoattraction of macrophage to remove the dead thymocytes.

IL-23 induces apoptosis of self-reactive thymocytes in thymic negative selection by an Rorc dependent mechanism (64.19)

Abstract Thymocyte apoptosis can occur by over-induction of a specific transcription factor, RORγt (or its gene, Rorc); however, the role of RORγt in thymocyte selections and the upstream signal that drives the expression of Rorc are largely unknown. We have observed that interleukin-23 (IL-23), a cytokine which has been implicated as a key regulator of Rorc, was expressed by thymic dendritic cells. To determine if the IL-23-RORγt signaling axis is involved in thymocyte selections, we have determined the expression of Il23r and Rorc in thymocytes from the male antigen-specific Db/H-Y T-cell receptor (TCR) transgenic (Tg) and the MOG35-55 antigen-specific 2D2 TCR Tg mice. Specific antigen activation induced Il23r expression on CD4+CD8+ double positive (DP) thymocytes and then upregulated the expression of Rorc with IL-23 stimulation, leading to dramatic induction of DP thymocyte apoptosis in vitro in TCR Tg+ mice. In vivo over-expression of IL-23 in B6 mice by adenovirus (AdIL-23) efficiently depleted DP thymocytes by promoting apoptosis (from 1.5% to 10%) as determined by in situ TUNEL staining. This AdIL-23 induced thymocyte apoptosis was partially blocked in Rorc+/- mice. Consistent with these, there was an increased percent of Db/H-Y Tg+ thymocytes in Il23 p19-/- mice, compared with that in p19 intact male Db/H-Y Tg mice. Taken together, our work suggests an important role for the IL-23-RORγt axis in the induction of TCR-mediated apoptosis and negative selection in thymocytes.

Metalexyl fungicide induced oxidative stress and apoptosis in mouse thymus: the effect of antox

Metalaxyl is a benzenoid fungicide used to control soil-borne fungal diseases on fruits, cotton, soybean, peanuts, ornamental and grasses. The present work aims to evaluate the effect of oxidative stress on the induction of apoptosis in thymocytes of mice by metalaxyl. The work extends to study the possible role played by antox in minimising the toxicity of metalaxyl. Thymus of metalexyl-treated mice showed many histopathological alterations. There was a loss of differentiation between region of cortex and medulla, abnormal arrangement of lymphocytes and degeneration of the trabecula. Treatment with metalaxyl leads to an increase in apoptotic rate of thymocytes as detected by flow cytometry. Moreover, metalaxyle administration to animals increased significantly malondialdehyde and decreased catalase activity. The results showed that treating animals with metalaxyl followed by antox revealed an improvement in the histological changes observed in animals treated with metalaxyl alone. Moreover, antox treatment leads to a decrease in the percent of apoptotic cells. A decrease of malondialdehyde level and increase of catalase activity was recorded.

Apoptosis in immunocytes induced by several types of pesticides

Several types of pesticides, such as organophosphates and organochlorines, can induce thymocyte apoptosis, resulting in thymic atrophy and predisposing the highly sensitive fetal immune system to loss of tolerance to self-antigens and subsequent increased risk for autoimmune disease and allergies. In the studies here, mouse primary thymocytes and a human acute T-cell leukemia cell line (J45.01) were employed to examine potential thymocyte apoptosis induced by several types of chemicals, including several commonly-used pesticides. Thymocytes and J45.01 cells were treated for 4 or 8 hr with varying doses of metamidophos, parathion, PNMC, or methoxychlor; dexamethasone was used as a positive control. Apoptosis, cell viability, the proportion of Annexin-V+ cells, the activities of caspases 3/7, 8, and 9, and the levels of DNA fragmentation in both the J45.01 cells and thymocytes were then examined. The results here show that with both cell types, there was an increase in the proportion of annexin-V+ cells and levels of DNA fragmentation following exposure to parathion, PNMC, methoxychlor, or dexamethasone (positive control); however, the levels of sensitivity appeared to differ between the cell types. Furthermore, caspase-7 and -8 activities also differed between the J45.01 cells and thymocytes when treated with PNMC, methoxychlor, or dexamethasone. A more precise characterization of these inter-cellular differences is the logical next step in our studies of the effects of these (and other) pesticides on immune cell integrity. These specific types of follow-on mechanistic experiments are currently underway in our laboratories.

Thymus as a target tissue of glucocorticoid action: What are the consequences of glucocorticoids thymectomy?

Glucocorticoids represent the most powerful endogenous anti-inflammatory and immunosuppressive effectors, interfering with virtually every step of immunoinflammatory responses. Glucocorticoids are often the most effective therapy in the prevention or suppression of inflammation and other immunologically mediated processes, but their use is limited by systemic side effects induced by the over-production of reactive oxygen species, causing dysregulation of physiological processes. The thymus is an organ with both endocrine and immune functions. Glucocorticoids induce thymocyte apoptosis, causing a profound reduction in thymic mass and volume and inducing hormonal thymectomy. The clinical aspects of glucocorticoid thymectomy are not under enough investigation. These unwanted systemic side effects may be the consequence of prolonged therapeutic application of glucocorticoids and prolonged or chronic activation of the hypothalamic-pituitary adrenal axis, which may lead to increased and prolonged secretion of glucocorticoids. This review will discuss the metabolic effects of glucocorticoids in the context of thymic physiology asthe primary sex hormone-responsive organ.

MerTK regulates thymic selection of autoreactive T cells

T cell-mediated autoimmune diseases such as type 1 diabetes (T1D) are believed to be the result in part of inefficient negative selection of self-specific thymocytes. However, the events regulating thymic negative selection are not fully understood. In the current study, we demonstrate that nonobese diabetic (NOD) mice lacking expression of the Mer tyrosine kinase (MerTK) have reduced inflammation of the pancreatic islets and fail to develop diabetes. Furthermore, NOD mice deficient in MerTK expression (Mer(-/-)) exhibit a reduced frequency of beta cell-specific T cells independent of immunoregulatory effectors. The establishment of bone marrow chimeric mice demonstrated that the block in beta cell autoimmunity required hematopoietic-derived cells lacking MerTK expression. Notably, fetal thymic organ cultures and self-peptide administration showed increased thymic negative selection in Mer(-/-) mice. Finally, thymic dendritic cells (DC) prepared from Mer(-/-) mice exhibited an increased capacity to induce thymocyte apoptosis in a peptide-specific manner in vitro. These findings provide evidence for a unique mechanism involving MerTK-mediated regulation of thymocyte negative selection and thymic DC, and suggest a role for MerTK in contributing to beta cell autoimmunity.

Involvement of cPLA2Inhibition in Dexamethasone-Induced Thymocyte Apoptosis

Various molecular mechanisms have been suggested to be involved in dexamethasone induced thymocyte apoptosis. In this study we show that pharmacological inhibition of cytoplasmic PLA2 in mouse thymocytes for 18 h with arachidonyl trifluoromethyl ketone (AACOCF3) (10 microM) and palmitoyl trifluoromethyl ketone (PACOCF3) (10 microM) induced a drastic increase of thymocyte apoptosis comparable to that observed following Dex (10(-7) M) treatment, while inhibition of secretory PLA2 with p-bromophenacyl bromide (pBPB) (20 microM) did not. AACOCF3-induced thymocyte apoptosis, similarly to Dex-induced thymocyte apoptosis, was eliminated by cell pre-treatment with the PI-PLCbeta inhibitor, U73122, but not by the PC-PLC inhibitor D609. These observations were corroborated by the ability of AACOCF3, like Dex, to induce a rapid and transient increase in DAG generation. In addition, AACOCF3-induced apoptosis involved the activation of the acidic sphingomyelinase (aSMase) but not of the neutral sphingomyelinase (nSMase), as evaluated by measurements of enzyme activity in cell extracts following thymocyte exposure to AACOCF3 and by the ability of monensin to inhibit AACOCF3-induced thymocyte apoptosis. In addition, the AACOCF3 apoptotic effect resulted in an early increase of ceramide levels. AACOCF3-induced thymocyte apoptosis involved the activation of caspase 3, and cell pre-treatment with a caspase 3 inhibitor prevented AACOCF3-induced apoptosis. These observations suggest that cPLA2 inhibition may have a role in Dex-induced thymocyte apoptosis and highlight the importance of cPLA2 activity in thymocyte survival.

A Role for the Fas/FasL System in Modulating Genetic Susceptibility to T-Cell Lymphoblastic Lymphomas

The Fas/FasL system mediates induced apoptosis of immature thymocytes and peripheral T lymphocytes, but little is known about its implication in genetic susceptibility to T-cell malignancies. In this article, we report that the expression of FasL increases early in all mice after gamma-radiation treatments, maintaining such high levels for a long time in mice that resisted tumor induction. However, its expression is practically absent in T-cell lymphoblastic lymphomas. Interestingly, there exist significant differences in the level of expression between two mice strains exhibiting extremely distinct susceptibilities that can be attributed to promoter functional polymorphisms. In addition, several functional nucleotide changes in the coding sequences of both Fas and FasL genes significantly affect their biological activity. These results lead us to propose that germ-line functional polymorphisms affecting either the levels of expression or the biological activity of both Fas and FasL genes could be contributing to the genetic risk to develop T-cell lymphoblastic lymphomas and support the use of radiotherapy as an adequate procedure to choose in the treatment of T-cell malignancies.