Multiple sclerosis retrovirus-like envelope gene: Role of the chromosome 20 insertion.

BACKGROUND: The genetic basis involved in multiple sclerosis (MS) susceptibility was not completely revealed by genome-wide association studies. Part of it could lie in repetitive sequences, as those corresponding to human Endogenous Retroviruses (HERVs). Retrovirus-like particles were isolated from MS patients and the genome of the MS-associated retrovirus (MSRV) was the founder of the HERV-W family. We aimed to ascertain which chromosomal origin encodes the pathogenic ENV protein by genomic analysis of the HERV-W insertions. METHODS/RESULTS: In silico analyses allowed to uncover putative open reading frames containing the specific sequence previously reported for MSRV-like envelope (env) detection. Out of the 261 genomic insertions of HERV-W env, only 9 copies harbor the specific primers and probe featuring MSRV-like env. The copy from chromosome 20 was further studied considering its size, a truncated homologue of the functional HERV-W env sequence encoding syncytin. High Resolution Melting analysis of this sequence identified two single nucleotide polymorphisms, subsequently genotyped by Taqman chemistry in 668 MS patients and 678 healthy controls. No significant association of these polymorphisms with MS risk was evidenced. Transcriptional activity of this MSRV-like env copy was detected in peripheral blood mononuclear cells from patients and controls. RNA expression levels of chromosome 20-specific MSRV-like env did not show significant differences between MS patients and controls, neither were related to genotypes of the two mentioned polymorphisms. CONCLUSIONS: The lack of association with MS risk of the identified polymorphisms together with the transcription results discard chromosome 20 as genomic origin of MSRV-like env.

HERV-W polymorphism in chromosome X is associated with multiple sclerosis risk and with differential expression of MSRV.

BACKGROUND: Multiple Sclerosis (MS) is an autoimmune demyelinating disease that occurs more frequently in women than in men. Multiple Sclerosis Associated Retrovirus (MSRV) is a member of HERV-W, a multicopy human endogenous retroviral family repeatedly implicated in MS pathogenesis. MSRV envelope protein is elevated in the serum of MS patients and induces inflammation and demyelination but, in spite of this pathogenic potential, its exact genomic origin and mechanism of generation are unknown. A possible link between the HERV-W copy on chromosome Xq22.3, that contains an almost complete open reading frame, and the gender differential prevalence in MS has been suggested. RESULTS: MSRV transcription levels were higher in MS patients than in controls (U-Mann-Whitney; p = 0.004). Also, they were associated with the clinical forms (Spearman; p = 0.0003) and with the Multiple Sclerosis Severity Score (MSSS) (Spearman; p = 0.016). By mapping a 3 kb region in Xq22.3, including the HERV-W locus, we identified three polymorphisms: rs6622139 (T/C), rs6622140 (G/A) and rs1290413 (G/A). After genotyping 3127 individuals (1669 patients and 1458 controls) from two different Spanish cohorts, we found that in women rs6622139 T/C was associated with MS susceptibility: [χ2; p = 0.004; OR (95% CI) = 0.50 (0.31-0.81)] and severity, since CC women presented lower MSSS scores than CT (U-Mann-Whitney; p = 0.039) or TT patients (U-Mann-Whitney; p = 0.031). Concordantly with the susceptibility conferred in women, rs6622139*T was associated with higher MSRV expression (U-Mann-Whitney; p = 0.003). CONCLUSIONS: Our present work supports the hypothesis of a direct involvement of HERV-W/MSRV in MS pathogenesis, identifying a genetic marker on chromosome X that could be one of the causes underlying the gender differences in MS.

Superantigen-primed T cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) replicate poorly following recall encounter.

The current study investigated the effect of tetrachlorodibenzo-p-dioxin (TCDD) on the ability of staphylococcal enterotoxin A (SEA)-primed T cells to divide by dual-labeling the cells with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) and antibodies against the specific T cell receptors. C57BL/6 wild-type mice were injected ip with TCDD (10 microg/kg body weight) followed by hind footpad injections of SEA (10 microg/footpad). The draining popliteal lymph nodes (PLN) were harvested 1-4 days posttreatment, labeled with CFSE and cultured for 1-4 days without further stimulation or in the presence of the recall antigen. TCDD-exposed SEA-reactive Vbeta3+ and Vbeta11+ T cells showed decreased cell divisions upon in vitro culture in the absence of any stimulation, which correlated with increased levels of apoptosis. The recall cell-division response was also defective in SEA-reactive T cells isolated from TCDD-exposed mice. However, during the recall response, cells from TCDD-exposed mice did not exhibit a defect in apoptosis, suggesting the defective recall response may result from a state of anergy rather than increased apoptosis. Using AhR knockout (KO) mice, we found AhR involvement in the regulation of defective cell division and apoptosis induced by TCDD. Together, these data demonstrate, while TCDD-induced apoptosis may account for the decreased primary T cell proliferative response, that the reduced cell division seen during subsequent exposure to recall antigen may result from a state of anergy. The study also demonstrates that a combined use of superantigen and CFSE may offer a simple and useful tool to monitor the ability of immunotoxicants to alter the proliferative responsiveness of antigen-specific T cells.

Treatment of mice with 2,3,7,8-tetrachlorodibenzo-p-dioxin leads to aryl hydrocarbon receptor-dependent nuclear translocation of NF-kappaB and expression of Fas ligand in thymic stromal cells and consequent apoptosis in T cells.

We investigated the role of aryl hydrocarbon receptor (AhR) in the regulation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced apoptosis in thymic T cells. AhR knockout (KO) mice were resistant to TCDD-induced thymic atrophy and apoptosis when compared with the AhR wild-type mice. TCDD triggered the expression of several apoptotic genes, including FasL in AhR wild-type but not AhRKO mice. TCDD-induced increase in FasL was seen only in thymic stromal but not thymic T cells. When TCDD-exposed stromal cells were mixed with untreated thymic T cells, increased apoptosis was detected in T cells that involved Fas-FasL interactions. Thus, apoptosis in T cells was not detected when TCDD-treated stromal cells from FasL-defective or AhRKO mice were mixed with wild-type T cells or when TCDD-exposed wild-type stromal cells were mixed with Fas-deficient T cells. TCDD treatment, in vivo and in vitro, led to colocalization and translocation of NF-kappaB subunits (p50, p65) to the nucleus in stromal but not T cells from AhR wild-type mice. NF-kappaB activation was not observed in stromal cells isolated from TCDD-treated AhRKO mice. Mutations in NF-kappaB-binding sites on the FasL promoter showed that TCDD regulates FasL promoter activity through NF-kappaB. TCDD treatment in vivo caused activation of the death receptor and mitochondrial pathways of apoptosis. Cross-talk between the two pathways was not necessary for apoptosis inasmuch as TCDD-treated Bid KO mice showed thymic atrophy and increased apoptosis, similar to the wild-type mice. These findings demonstrate that AhR regulates FasL and NF-kappaB in stromal cells, which in turn plays a critical role in initiating apoptosis in thymic T cells.

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on maternal immune response during pregnancy.

Whether pregnancy-induced immunosuppression when combined with exposure to 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) could exacerbate immunotoxicity has not been previously investigated. The current study evaluated the immune status of C57BL/6 pregnant and virgin mice following exposure to TCDD. To this end, syngeneically pregnant or virgin female mice were injected intraperitoneally with 10 micro g/kg TCDD. Pregnancy alone significantly decreased thymic cellularity and J11d expression as well as induced changes in T-cell subsets. TCDD treatment caused significant thymic atrophy in pregnant mice as early as 48 h after exposure, but this effect was apparent in virgin mice only after 72 h. TCDD treatment also caused more marked alterations in thymic T-cell subpopulations of pregnant mice when compared to the virgin mice, with a decrease in the percentage of double-positive T cells and an increase in the percentage of single-positive (sP CD4(+) or sP CD8(+)) and double-negative T cells. Moreover, the proliferative responses of thymocytes, but not splenocytes, to mitogens were significantly altered in TCDD-treated pregnant mice when compared to the TCDD-treated virgin mice. Furthermore, no significant changes in the expression of CD4, CD8, B220 and NK1.1 markers were found in splenocytes from TCDD-treated virgin and pregnant mice. Immunization of mice with a superantigen caused a similar immunotoxic response in TCDD-treated pregnant and virgin mice with a decreased lymph node cellularity and lower percentages and cell numbers of Vbeta3(+) and Vbeta11(+) T cells. Together, the results of the current study demonstrate for the first time that pregnancy augments the sensitivity to TCDD-induced immunotoxicity in the thymus, but not in secondary lymphoid organs.

Evidence for induction of apoptosis in T cells from murine fetal thymus following perinatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

Perinatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes thymic atrophy, but the precise mechanism of such toxicity remains unresolved. The current study investigated the role of apoptosis in TCDD-induced thymic involution following perinatal exposure to TCDD. To this end, C57BL/6 pregnant mice were injected intraperitoneally on gestational day (GD) 14 with a single dose of 10 microg/kg TCDD. Analysis of the thymus on GDs 15, 16, 17, and 18, and on postnatal day (PD) 1, showed a remarkable reduction in thymic cellularity 3-7 days post-TCDD exposure. TCDD treatment also caused marked changes in the proportions of T-cell subsets, particularly on GD 17 and GD 18 thymocytes. In vitro culture of thymocytes from mice exposed perinatally to TCDD showed increased apoptosis when compared to the controls, which peaked on day 3 post-TCDD exposure. Triple-color staining showed that TCDD induced apoptosis in all four subpopulations of T cells, with the double-positive T cells undergoing the highest level. Moreover, increased cleavage of caspase-3 was seen when TCDD-exposed GD 17 thymocytes were directly tested. Furthermore, apoptosis-associated phenotypic changes were found in thymocytes of mice perinatally exposed to TCDD, characterized by an increase in expression of CD3, alphabetaTCR, IL-2R, and CD44, and a decrease in CD4, CD8, and J11d markers. Finally, thymocytes from mice exposed perinatally to TCDD showed higher levels of Fas, TRAIL, and DR5 mRNA, but the levels of Bcl-2, Bcl-xL, and Bax were either unaltered or changed moderately. Taken together, these results suggest that TCDD-induced thymic atrophy following perinatal exposure may result, at least in part, from increased apoptosis mediated by death receptor pathway involving Fas, TRAIL, and DR5.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces Fas-dependent activation-induced cell death in superantigen-primed T cells.

Immune response against a foreign antigen is characterized by a growth phase, in which antigen-specific T cells clonally expand, followed by a decline phase in which the activated T cells undergo apoptosis, a process termed activation-induced cell death (AICD). In the current study, we have investigated the phase at which 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) acts to downregulate the antigen-specific T cell response. To this end, C57BL/6 +/+ mice were injected with staphylococcal enterotoxin A (SEA) into the footpads (10 micro g/footpad), and simultaneously treated with TCDD (10 or 50 micro g/kg intraperitoneally). At various time points, the draining lymph node (LN) cells were analyzed for SEA-activated T cells. The data demonstrated that in C57BL/6 +/+ mice, TCDD treatment did not alter the growth phase but facilitated the decline phase of SEA-reactive T cells. TCDD caused a significant decrease in the percentage and absolute numbers of CD4(+) and CD8(+) SEA-responsive T cells expressing Vbeta3(+) and Vbeta11(+) but did not affect SEA-nonresponsive Vbeta8(+) T cells. Upon in vitro culture, TCDD-exposed SEA-immunized LN cells exhibited increased levels of apoptosis when compared with the vehicle controls. When Fas-deficient (C57BL/6 lpr/lpr) or Fas ligand defective (C57BL/6 gld/gld) mice were treated with TCDD, they failed to exhibit a decrease in percentage and cellularity of SEA-reactive T cells, thereby suggesting a role of Fas-Fas ligand interactions in the TCDD-induced downregulation of SEA-reactive T cell response. The resistance to TCDD-induced decrease in T cell responsiveness to SEA seen in Fas- and FasL-mutant mice was neither due to decreased aryl hydrocabon receptor (AhR) expression nor to altered T cell responsiveness to SEA. The current study demonstrates that TCDD does not prevent T cell activation, but prematurely induces Fas-based AICD, which may contribute to the deletion of antigen-primed T cells.

Analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced gene expression profile in vivo using pathway-specific cDNA arrays.

In the current study, we used pathway-specific cDNA arrays to detect the transcriptional signature induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in vivo by studying simultaneously the expression profiles of 83 genes involved in apoptosis, cytokine production and angiogenesis. To this end, C57BL/6 mice were injected i.p. with 50 microg/kg body weight of TCDD and 1 or 3 days later, the thymus was analyzed for gene expression profiles. In the thymus, 23 out of 37 apoptotic genes screened were up-regulated by TCDD by a factor of two or more when compared to the vehicle-treated controls. In contrast, in the spleen, 20 out of 22 and in the liver, 16 out of 37 apoptotic genes were up-regulated. In the thymus, several genes encoding caspases, and members of the TNF family, including Fas ligand, were induced. Also, in the thymus, eight out of 23, and in the spleen, six out of 23 cytokine genes were up-regulated. In the liver and to a lesser extent in the thymus, certain angiogenesis genes were induced while others were repressed. When mice were injected with 0.1, 1, 10 or 50 microg/kg body weight of TCDD and the thymus was analyzed for apoptotic genes 1 day later, a dose-dependent response was not seen with most apoptotic genes. However, certain apoptotic genes were induced in the thymus even at low doses of 0.1 microg/kg body weight of TCDD. These data demonstrate that TCDD alters the expression of a large array of genes involved in apoptosis, cytokine production and angiogenesis. Thus, pathway-specific cDNA arrays may help in the identification of specific gene expression profiles induced by xenobiotics and to delineate the molecular mechanisms of toxicity.