α7 nAChR mediated Fas demethylation contributes to prenatal nicotine exposure-induced programmed thymocyte apoptosis in mice.

This study aimed to investigate the effects of prenatal nicotine exposure (PNE) on thymocyte apoptosis and postnatal immune impairments in vivo and further explore the epigenetic mechanisms of the pro-apoptotic effect of nicotine in vitro. The results showed that PNE caused immune impairments in offspring on postnatal day 49, manifested as increased IL-4 production and an increased IgG1/IgG2a ratio in serum. Enhanced apoptosis of total and CD4+SP thymocytes was observed both in fetus and in offspring. Further, by exposing thymocytes to 0-100 µM of nicotine in vitro for 48 h, we found that nicotine increased α7 nicotinic acetylcholine receptor (nAChR) expression, activated the Fas apoptotic pathway, and promoted thymocyte apoptosis in concentration-dependent manners. In addition, nicotine could induce Tet methylcytosine dioxygenase (TET) 2 expression and Fas promoter demethylation, which can be abolished by TET2 siRNA transfection. Moreover, the α7 nAChR specific antagonist α-bungarotoxin can abrogate nicotine-induced TET2 increase, and the following Fas demethylation and Fas-mediated apoptosis. In conclusion, our findings showed, for the first time, that α7 nAChR activation could induce TET2-mediated Fas demethylation in thymocytes and results in the upregulation of Fas apoptotic pathway, which provide evidence for elucidating the PNE-induced programmed thymocyte apoptosis.

The regulation of the mitochondrial apoptotic pathway by glucocorticoid receptor in collaboration with Bcl-2 family proteins in developing T cells.

Glucocorticoids (GC) are important in the regulation of selection and apoptosis of CD4+CD8+ double-positive (DP) thymocytes. The pronounced GC-sensitivity of DP thymocytes, observed earlier, might be due to the combination of classical (genomic) and alternative (non-genomic) glucocorticoid receptor (GR) signaling events modifying activation or apoptotic pathways. In particular, the previously demonstrated mitochondrial translocation of activated GR in DP thymocytes offered a fascinating explanation for their pronounced GC-induced apoptosis sensitivity. However, the fine molecular details how the mitochondrial translocation of GR might regulate apoptosis remained unclear. Therefore, in the present study, we intended to examine which apoptotic pathways could be involved in GC-induced thymocyte apoptosis. Furthermore we investigated the potential relationship between the GR and Bcl-2 proteins. Using an in vitro test system, thymocytes from 4-week-old BALB/c mice, were treated with the GC-analogue dexamethasone (DX). Bax accumulated in mitochondria upon DX treatment. Mitochondrial GR showed association with members of the Bcl-2 family: Bak, Bim, Bcl-xL. Elevated Cytochrome C, and active caspase-3, -8, and -9 levels were detected in thymocytes after DX treatment. These results support the hypothesis that in early phases of GC-induced thymocyte apoptosis, the mitochondrial pathway plays a crucial role, confirmed by the release of Cytochrome C and the activation of caspase-9. The activation of caspase-8 was presumably due to cross-talk between apoptotic signaling pathways. We propose that the GC-induced mitochondrial accumulation of Bax and the interaction between the GR and Bim, Bcl-xL and Bak could play a role in the regulation of thymocyte apoptosis.

Transcriptomic analysis reveals sex-specific differences in the expression of Dcl1 and Fis1 genes in the radio-adaptive response of thymocytes to TRP53-mediated apoptosis.

BACKGROUND: Radio-Adaptive Response (RAR) is a biological defense mechanism whereby exposure to low dose ionizing radiation (IR) mitigates the detrimental effects of high dose irradiation. RAR has been widely observed in vivo using as endpoint less induction of apoptosis. However, sex differences associated with RAR and variations between males and females on global gene expression influenced by RAR have not been still investigated. In addition, the response to radiation-induced apoptosis is associated with phosphorylation of TRP53 at both the serine 15 (ser-18 in the mouse) and serine 392 (ser-389 in mice) residues, but the role of these two phosphorylated forms in male and female RAR remains to be elucidated. RESULTS: We analyzed the effect of administering priming low dose radiation (0.075 Gy of X-rays) prior to high dose radiation (1.75 Gy of γ-rays) on the level of caspase-3-mediated apoptosis and on global transcriptional expression in thymocytes of male and female mice. Here, we provide the first evidence of a differential sex effect of RAR on the reduction of thymocyte apoptosis with males showing lesser levels of caspase-3-mediated apoptosis than females. Analysis of transcriptomic profiles of 1944 genes involved in apoptosis signaling in radio-adapted thymocytes identified 17 transcripts exhibiting differential expression between both sexes. Among them, Dlc1 and Fis1 are closely related to the apoptosis mediated by the TRP53 protein. Our data demonstrate that overexpression of Dlc1 and Fis1 occur concomitantly with a highest accumulation of phosphoserine-18-TRP53 and caspase-3 in radio-adapted thymocytes of female mice. In an opposite way, both down-modulation of Fis1 and phosphoserine-389-TRP53 accumulation appear to be associated with protection from thymocyte apoptosis mediated by caspase-3 in males. CONCLUSIONS: Transcriptomic analysis performed in this work reveals for the first time sex-specific differences in gene expression influenced by RAR. Our results also suggest a sex-dependent dual role for phosphoserine-18-TRP53 and phosphoserine-389-TRP53 in the regulation of the radio-adaptive response in mouse thymocytes.

Nitroxidergic modulation of behavioural, cardiovascular and immune responses, and brain NADPH diaphorase activity upon morphine tolerance/dependence in rats.

Opioid and non-opioid effects of acute and chronic morphine administration on behaviour, cardiovascular responses, cell proliferation and apoptosis and nitric-oxide synthase (NOS) activity were studied in rats. A novel score-point scale was introduced to quantify the signs of opioid withdrawal syndrome. NOS inhibitor L-NAME (NG-nitro-L-arginine methyl ester) was applied to reveal the role of NOS/NO pathway in the modulation of morphine-induced in vivo and in vitro responses. The obtained data showed that chronic co-administration of L-NAME drastically attenuated naloxone-precipitated withdrawal syndrome and prevented the development of morphine tolerance to cardiovascular action of morphine. The apoptotic process was very much restricted by L-NAME supplementation of chronic morphine treatment, which resulted in few apoptotic cells, less low molecular weight genomic DNA and preservation of high molecular weight non-fragmented genomic DNA. The study provides new data for nitroxidergic modulation of opioid tolerance and dependence.

Effects of catecholamines on thymocyte apoptosis and proliferation depend on thymocyte microenvironment.

The present study, through quantification of tyrosine hydroxylase (TH) expression and catecholamine (CA) content in the presence and in the absence of α-methyl-p-tyrosine (AMPT), a TH inhibitor, in adult thymic organ (ATOC) and thymocyte culture, demonstrated that thymic cells produce CAs. In addition, in ATOC an increase in ß2-adrenoceptor (AR) mRNA expression and ß2-AR thymocyte surface density was registered. Furthermore, AMPT (10(-4)M), as propranolol (10(-4)M), augmented thymocyte apoptosis and diminished thymocyte proliferation in ATOC. Propranolol exerted these effects acting on CD3(high) thymocytes. However, in thymocyte cultures, propranolol (10(-6)M) acting on the same thymocyte subset exerted the opposing effect on thymocyte apoptosis and ConA-stimulated proliferation. This suggested that, depending on thymocyte microenvironment, differential effects can be induced through the same type of AR. Additionally, arterenol (10(-8) to 10(-6)M), similar to propranolol, diminished apoptosis, but increased ConA-stimulated thymocyte proliferation in thymocyte culture. However, differently from propranolol, arterenol affected manly CD3- thymocyte subset, which harbors majority of α1-AR+thymocytes. Additionally, arterenol showed a dose-dependent decrease in efficiency of thymocyte apoptosis and proliferation modulation with the rise in its concentration. Considering greater affinity of arterenol for α1-ARs than for ß2-ARs, the previous findings could be attributable to increased engagement of ß2-ARs with the rise of arterenol concentration. Consistently, in the presence of propranolol (10(-6)M), a ß-AR blocker, the arterenol (10(-8)M) effects on thymocytes were augmented. In conclusion, thymic endogenous CAs, acting through distinct AR types and, possible, the same AR type (but in different cell microenvironment) may exert the opposing effects on thymocyte apoptosis/proliferation.

An anti-DEC-205 monoclonal antibody stimulates binding of thymocytes to rat thymic dendritic cells and promotes apoptosis of thymocytes.

DEC-205, a transmembrane receptor responsible for cross-presentation of apoptotic cell-derived antigens, is expressed by cortical thymic epithelial cells (TEC) and thymic dendritic cells (TDC) in humans and mice, but its function in T-cell development is still unclear. In this work we have studied for the first time the expression of DEC-205 in the rat thymus by HD83 monoclonal antibody (mAb) and immunohistochemistry, as well as the ability of this mAb to modulate thymocyte - TDC interactions in vitro. We showed the positivity of cortical TEC in situ, including thymic nurse cells (TNC) in suspension, and TDC, whereas subcapsular, perivascular and medullary TEC were negative. All examined DEC-205 positive and DEC-205 negative structures were MHC class II positive. HD83 mAb increased apoptosis of thymocytes in co-culture with TDC in vitro and the process was associated with increased binding of thymocytes to TDC in a rosette form. Since negative selection of thymocytes by clonal deletion (apoptosis) was mediated predominantly by TDC, our results suggest the possible indirect effect of the DEC-205 molecule in these mechanisms.

Glucocorticoids, master modulators of the thymic catecholaminergic system?

There is evidence that the major mediators of stress, i.e., catecholamines and glucocorticoids, play an important role in modulating thymopoiesis and consequently immune responses. Furthermore, there are data suggesting that glucocorticoids influence catecholamine action. Therefore, to assess the putative relevance of glucocorticoid-catecholamine interplay in the modulation of thymopoiesis we analyzed thymocyte differentiation/maturation in non-adrenalectomized and andrenalectomized rats subjected to treatment with propranolol (0.4 mg·100 g body weight-1·day-1) for 4 days. The effects of β-adrenoceptor blockade on thymopoiesis in non-adrenalectomized rats differed not only quantitatively but also qualitatively from those in adrenalectomized rats. In adrenalectomized rats, besides a more efficient thymopoiesis [judged by a more pronounced increase in the relative proportion of the most mature single-positive TCRαβhigh thymocytes as revealed by two-way ANOVA; for CD4+CD8- F (1,20) = 10.92, P < 0.01; for CD4-CD8+ F (1,20) = 7.47, P < 0.05], a skewed thymocyte maturation towards the CD4-CD8+ phenotype, and consequently a diminished CD4+CD8-/CD4-CD8+ mature TCRαβhigh thymocyte ratio (3.41 ± 0.21 in non-adrenalectomized rats vs 2.90 ± 0.31 in adrenalectomized rats, P < 0.05) were found. Therefore, we assumed that catecholaminergic modulation of thymopoiesis exhibits a substantial degree of glucocorticoid-dependent plasticity. Given that glucocorticoids, apart from catecholamine synthesis, influence adrenoceptor expression, we also hypothesized that the lack of adrenal glucocorticoids affected not only β-adrenoceptor- but also α-adrenoceptor-mediated modulation of thymopoiesis.

Peripubertal orchidectomy transitorily affects age-associated thymic involution in rats

The role of gonadal hormones in induction and, particularly, maintenance/progression of rat thymic involution, which normally starts around puberty, was reassessed by examining the effects of peripubertal orchidectomy on thymic weight and morphometric parameters at different times up to the age of 10 months. Up to 6 months post-castration both thymic weight and cellularity in orchidectomized (Cx) rats were greater than in age-matched control rats, sham Cx (Sx). The increase in thymic cellularity reflected an increase in thymocyte proliferation rate (the proportion of proliferating cells was 18.6 ± 0.7 percent in 2-month-old Cx (N = 5) vs 13.4 ± 0.3 percent (N = 5) in age-matched Sx rats) followed by reduced sensitivity to apoptotic signals (apoptotic thymocytes were 9.8 ± 0.9 percent in 2-month-old Cx (N = 5) vs 15.5 ± 0.3 percent (N = 5) age-matched Sx rats). However, 9 months post-orchidectomy, neither thymic weight and cellularity nor any of the morphometric parameters analyzed differed between Cx and control rats. The reduction of thymic cellularity in Cx rats to control values may be related to increased sensitivity of their thymocytes to apoptotic signals in culture (72.6 ± 1.2 percent in 10-month-old vs 9.8 ± 0.9 percent in 2-month-old Cx rats) followed by reduced responsiveness to proliferative stimuli (14.1 ± 0.2 percent in 10-month-old vs 18.6 ± 0.7 percent in 2-month-old Cx rats). Thus, the study indicates that the effects of peripubertal orchidectomy on thymic weight and cellularity, as well as on the main morphometric indices, are long-lasting but not permanent, i.e., that removal of the testes can only postpone but not prevent age-related organ atrophy and consequently functional deterioration of the immune system.