Tumor Necrosis Factor Receptor 2 Expression Research Articles

Clinical implications of tumor necrosis factor receptor 2 in breast cancer.

Tumor necrosis factor receptor 2 (TNFR2) is a member of the tumor necrosis factor receptor family. Its high expression and oncogenic roles have been reported in several types of tumors in previous years. However, the clinical implication of TNFR2 in breast cancer (BC) tissue (i.e., not soluble TNFR2 in blood or genetic variation of TNFR2) has not been reported. In the present study, TNFR2 expression was detected in BC tissue using immunohistochemistry and, to the best of our knowledge, it was confirmed for the first time that TNFR2 was positively associated with increased tumor size, advanced clinical stage and higher pathological grade. Survival analysis revealed that TNFR2 was positively associated with shorter overall survival (OS) time and disease-free survival (DFS) time. In addition, univariate regression analysis demonstrated that TNFR2 expression (P=0.045), tumor size (P<0.0001), clinical stage (P<0.0001), pathological grade (P=0.002), estrogen and progesterone receptor and human epidermal growth factor receptor 2 (HER2) triple-status (P=0.001) all had a significant impact on the OS rate of patients with BC. TNFR2 expression (P=0.017), age (P=0.011), menopausal status (P<0.0001), tumor size (P=0.016), clinical stage (P=0.005), pathological grade (P=0.002) and estrogen/progesterone receptor and HER2 triple-status (P=0.008) were all shown to significantly impact the DFS rate of patients with BC. Multivariate regression analysis showed that only clinical stage (P=0.024), estrogen and progesterone receptor status and HER2 status (P=0.009) had a significant impact on the OS rate of patients with BC, while TNFR2 expression (P=0.043) and menopausal status (P=0.033) were shown to significantly impact the DFS rate of patients with BC. These data indicated that TNFR2 may perform important roles in the progression and prognosis of BC. This enriches previous understanding about TNFR2 in BC.

TNFR2 promotes Adriamycin resistance in breast cancer cells by repairing DNA damage.

In recent years, tumor necrosis factor receptor2 (TNFR2) has attracted increasing attention for its important roles in promoting proliferation, migration and angiogenesis in several types of cancer. However, its role in drug resistance remain unclear. In the present study, TNFR2 expression levels in MDA‑MB‑231 and MCF‑7 breast cancer cells were demonstrated to be associated with Adriamycin (ADM) resistance. Silencing of TNFR2 in MCF‑7 cells significantly inhibited ADM resistance, while overexpression of TNFR2 in MDA‑MB‑231 cells significantly enhanced ADM resistance. ADM treatment induced phosphorylation of the histone family2A variant X (pH2AX), an established marker of DNA damage. Silencing of TNFR2 in MCF‑7 cells further induced pH2AX levels but inhibited the expression of the DNA damage repair protein, poly(ADP‑ribose) polymerase (PARP). By contract, overexpression of TNFR2 in MDA‑MB‑231 cells decreased pH2AX levels and enhanced PARP expression. Of note, treatment with the PARP inhibitor ABT888 significantly abrogated the effects of TNFR2 on pH2AX expression. On a molecular mechanism level, TNFR2 significantly affected the phosphorylation of AKT serine/threonine kinase 1 (AKT) in both cell lines, and treatment with the AKT inhibitor LY294002 effectively abrogated TNFR2‑induced PARP expression. Adrug resistance assay demonstrated that treatment with either LY294002 or ABT888 inhibited ADM resistance in breast cancer cells, and combination treatment with both LY294002 and ABT888 exhibited a significantly stronger inhibition effect on ADM resistance. The present results indicated that TNFR2 promoted ADM resistance in breast cancer cells by regulating the DNA damage repair protein PARP via the AKT signaling pathway.

Deep brain stimulation of the anterior nucleus of the thalamus reverses the gene expression of cytokines and their receptors as well as neuronal degeneration in epileptic rats

BackgroundDeep brain stimulation of the anterior nucleus of the thalamus (ANT-DBS) is effective in seizure control. However, the mechanisms remain unclear. MethodsSixty-four rats were randomly assigned to the control group, the kainic acid (KA) group, the sham-DBS group and the DBS group. Video-electroencephalogram (EEG) was used to monitor seizures. Quantitative real time PCR (qPCR) was applied for detecting interleukin-1 beta (IL-1β), IL-1 receptor (IL-1R), IL-6, IL-6 receptor (IL-6R), gp130, tumor necrosis factor-alpha (TNF-α), TNF-receptor 1 (TNF-R1) and TNF-receptor 2 (TNF-R2) expression 12h after the establishment of an epileptic model. The neuronal structural degeneration in the hippocampus was evaluated with transmission electron microscopy (TEM) at this same time point. ResultsThe seizure frequency was 48.6% lower in the DBS group compared with the sham-DBS group (P<0.01). The expression of IL-1β, IL-1R, IL-6, IL-6R, gp130, TNF-α and TNF-R1 was elevated in both the KA and the sham group compared with the control group (all Ps<0.01). Additionally, ANT-DBS was able to reverse this gene expression pattern in the DBS group compared with the sham-DBS group (all Ps<0.01). There was no significant difference in TNF-R2 expression among the four groups. The neuronal structural degeneration in the KA group and the sham-DBS group was more severe than that in the control group (injury scores, all Ps<0.01). ANT-DBS was also capable of relieving the degeneration compared with the sham-DBS group (injury score, P<0.01). ConclusionsThis study demonstrated that ANT-DBS can reduce seizure frequency in the early stage in epileptic rats as well as relieve the pro-inflammatory state and neuronal injury, which may be one of the most effective mechanisms of ANT-DBS against epileptogenesis.

Persistent astrocyte activation in the fragile X mouse cerebellum.

Fragile X Syndrome, the most common single gene cause of autism, results from loss of the RNA-binding protein FMRP. Although FMRP is highly expressed in neurons, it has also recently been identified in glia. It has been postulated that in the absence of FMRP, abnormal function of non-neuronal cells may contribute to the pathogenesis of the disorder. We previously demonstrated reduced numbers of oligodendrocyte precursor cells and delayed myelination in the cerebellum of fragile X (Fmr1) knockout mice. We used quantitative western blotting and immunocytochemistry to examine the status of astrocytes and microglia in the cerebellum of Fmr1 mice during development and in adulthood. We report increased expression of the astrocyte marker GFAP in the cerebellum of Fmr1 mice starting in the second postnatal week and persisting in to adulthood. At 2weeks postnatal, expression of Tumor Necrosis Factor Receptor 2 (TNFR2) and Leukemia Inhibitory Factor (LIF) were elevated in the Fmr1 KO cerebellum. In adults, expression of TNFR2 and the glial marker S100β were also elevated in Fmr1 knockouts, but LIF expression was not different from wild-type mice. We found no evidence of microglial activation or neuroinflammation at any age examined. These findings demonstrate an atypical pattern of astrogliosis in the absence of microglial activation in Fmr1 knockout mouse cerebellum. Enhanced TNFR2 and LIF expression in young mice suggests that changes in the expression of astrocytic proteins may be an attempt to compensate for delayed myelination in the developing cerebellum of Fmr1 mice.

A Gut Microbial Metabolite of Linoleic Acid, 10-Hydroxy-cis-12-octadecenoic Acid, Ameliorates Intestinal Epithelial Barrier Impairment Partially via GPR40-MEK-ERK Pathway

Gut microbial metabolites of polyunsaturated fatty acids have attracted much attention because of their various physiological properties. Dysfunction of tight junction (TJ) in the intestine contributes to the pathogenesis of many disorders such as inflammatory bowel disease. We evaluated the effects of five novel gut microbial metabolites on tumor necrosis factor (TNF)-α-induced barrier impairment in Caco-2 cells and dextran sulfate sodium-induced colitis in mice. 10-Hydroxy-cis-12-octadecenoic acid (HYA), a gut microbial metabolite of linoleic acid, suppressed TNF-α and dextran sulfate sodium-induced changes in the expression of TJ-related molecules, occludin, zonula occludens-1, and myosin light chain kinase. HYA also suppressed the expression of TNF receptor 2 (TNFR2) mRNA and protein expression in Caco-2 cells and colonic tissue. In addition, HYA suppressed the protein expression of TNFR2 in murine intestinal epithelial cells. Furthermore, HYA significantly up-regulated G protein-coupled receptor (GPR) 40 expression in Caco-2 cells. It also induced [Ca(2+)]i responses in HEK293 cells expressing human GPR40 with higher sensitivity than linoleic acid, its metabolic precursor. The barrier-recovering effects of HYA were abrogated by a GPR40 antagonist and MEK inhibitor in Caco-2 cells. Conversely, 10-hydroxyoctadacanoic acid, which is a gut microbial metabolite of oleic acid and lacks a carbon-carbon double bond at Δ12 position, did not show these TJ-restoring activities and down-regulated GPR40 expression. Therefore, HYA modulates TNFR2 expression, at least partially, via the GPR40-MEK-ERK pathway and may be useful in the treatment of TJ-related disorders such as inflammatory bowel disease.

TNFR2 Is Expressed Preferentially By Late Differentiated CD8 T-Cells and Can be Triggered By TNFR2-Specific Ligand to Induce Cell Death of Recently Activated Antigen-Specific T Cells: A Possible Role of TNFR2 in T-Cell Deflation

Recent evidence suggests that Tumor Necrosis Factor (TNF) can selectively kill antigen-specific autoreactive CD8+ T-cells through engagement with TNF Receptor 2 (TNFR2) (1). Within the immune system, TNFR2 expression is restricted to subsets of T-cells, a profile which is in marked contrast to the ubiquitous pattern of expression of TNFR1. However, the spectrum and physiological significance of TNFR2 expression by CD8+ T-cell subpopulations is unknown.In this study we analysed the expression of TNFR2 by CD8 T-cell subsets isolated from normal healthy donors by flow cytometry. In addition, in order to understand the physiological significance of TNFR2 expression on recently activated T cells, we further studied expression on CMV-specific CD8 T-cells which expanded in stem cell transplant patients in response to episodes of CMV reactivation. The expression of TNFR2 was compared to that of other common gamma chain receptors including IL2R and IL7R, and to the expression of a receptor for inflammatory cytokine IL6.TNFR2 expression was found to increase during differentiation of CD8+ T cells. In particular, TNFR2 expression was seen on 6.5% of naïve, 14.6% of central memory, 37.9% of effector memory and 45.2% of CD45RA-revertant effector memory (TEMRA) CD8+ T cells. In contrast, common gamma chain cytokine receptor expression was skewed towards less differentiated T-cell subsets. For example, IL-7R was expressed by 63% of central memory populations but only 18.4% of the TEMRA subset. Comparable expression of IL2R was 12.1% on TCM and 2% on TEMRA. Of interest, IL-6 receptor expression was predominantly expressed by naïve CD8 T-cells (69.5%). In support of these results, we went on to show that expression of TNFR2 was inducible on primary T cells following activation with anti-CD3 and IL-2 in vitro. Healthy CMV seropositive donors had a larger median number of CD8+ T cells expressing TNFR2 (53%) in comparison to CMV seronegative donors (15%), (p<0.0001), consistent with the known accumulation of differentiated T-cells within CMV seropositive individuals.The expression of TNFR2 was then examined on CMV-specific CD8 T-cells which were undergoing acute expansion in response to viremia in six haemopoietic stem cell transplant patients. The expansion of CMV-specific CD8 T-cells was accompanied by an increase in the intensity of TNFR2 expression which later decreased during the retraction of antigen-specific T-cells during resolution of viremia. In order to explore the functional significance of TNFR2 expression, T-cells isolated from healthy donors were treated with recombinant TNFR2-specific ligand. This induced cell loss ranging from 13% to 60% of all CD8 T-cells in relation to untreated control cells, with selective depletion of the TNFR2+ population. A similar proportion of CMV-specific T-cells from transplant patients were eliminated by ex vivo stimulation of TNFR2.In conclusion our work shows that TNFR2 expression increases during differentiation of CD8+ T cells. In addition, we were able to utilize virus-specific T cells from SCT patients to show that expression is increased during the acute response to stimulation with antigen. We also provide evidence that TNFR2 activation can lead to the partial elimination of antigen-specific CMV-specific T-cells and it may thus play an important role in the ‘deflation’ of a pathogen-specific T-cell immune response following resolution of infection. These data suggest that TNFR2 expression may act as a ligand to signal activation-induced cell death in late differentiated populations of CD8+ T cells. Further investigations are required to assess the molecular pathways of TNFR2 signalling that are activated following receptor ligation in vivoand whether or not these are disrupted in disorders associated with chronic CD8+ T cell lymphproliferation.(1) L. Ban et al, PNAS 2008, 105: 3644 DisclosuresNo relevant conflicts of interest to declare.

Maternal tumor necrosis factor receptor 2 gene variants associated with pre‐eclampsia in Tunisian women

The tumor necrosis factor receptor 2 (TNFR2) is expressed in placental tissue and it is involved in immune responses, inflammation, angiogenesis and blood pressure regulation; which makes it an attractive pre-eclampsia (PE) candidate gene. Furthermore, TNFR2 expression is altered in the first trimester in placentas of women who are destined to develop PE. Therefore, we examined the association between maternal and fetal genetic variants of TNFR2 and PE. Women with PE (n = 157) and their offspring with PE (n = 60) were compared to a control group of women (n = 97) and their offspring (n = 52) in the same Tunisian hospital-based population. We genotyped by polymerase chain reaction and restriction fragment length polymorphism the T/G polymorphism at position 676 in exon 6 (rs1061622) of the TNFR2 gene and examined its association with PE. The frequencies of TNFR2 (G/G) genotype and G allele were higher in the mothers with PE (n = 154) compared to the control group (15.3% vs 4.1% and 37% vs 26.3%, respectively); furthermore, the difference reached statistical significance (P = 0.002, odds ratio = 4.9; 95% confidence interval: 1.69-17.4 and P = 0.03, odds ratio = 1.69; 95% confidence interval: 1.03-2.8, respectively). In contrast, the fetal genotype and allele frequencies of this polymorphism had no effect on the risk of PE. The exon 6 polymorphism in TNFR2 (rs1061622) or a gene at proximity is associated specifically with PE at least in the Tunisian population and could increase the risk for PE for mothers carrying the homozygote minor allele. Nevertheless, these results need to be confirmed in other populations.

Tumor Necrosis Factor-α Antagonist Reduces Apoptosis of Neurons and Oligodendroglia in Rat Spinal Cord Injury

To examine the effects of a tumor necrosis factor (TNF)-α antagonist (etanercept) on rat spinal cord injury and identify a possible mechanism for its action. To elucidate the contribution of etanercept to the pathologic cascade in spinal cord injury and its possible suppression of neuronal and oligodendroglial apoptosis. Etanercept has been recently used successfully for treatment of inflammatory disorders. However, only a few studies have examined its role in suppressing neuronal and oligodendroglial apoptosis in spinal cord injury. Etanercept or saline (control) was administered by intraperitoneal injection 1 hour after thoracic spinal cord injury in rats. The expressions and localizations of TNF-α, TNF receptor 1 (TNFR1), and TNF receptor 2 (TNFR2) were examined by immunoblot and immunohistochemical analyses. Spinal cord tissue damage between saline- and etanercept-treated groups was also compared after hematoxylin-eosin and luxol fast blue (LFB) staining. The Basso-Beattie-Bresnahan (BBB) scale was used to evaluate rat locomotor function after etanercept administration. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells were counted and the immunoreactivity to active caspase-3 and caspase-8 was examined after etanercept administration. Immunoblot and double immunofluorescence staining revealed suppression of TNF-α, TNFR1, and TNFR2 expression after administration of etanercept in the acute phase of spinal cord injury. LFB staining demonstrated potential myelination in the etanercept-treated group from 2 week after spinal cord injury, together with an increased BBB locomotor score. Double immunofluorescence staining showed a significant decrease in TUNEL-positive neurons and oligodendroglia from 12 hour to 1 week in the gray and white matters after etanercept administration. Immunoblot analysis demonstrated overexpression of activated caspase-3 and caspase-8 after spinal cord injury, which was markedly inhibited by etanercept. Our results indicated that etanercept reduces the associated tissue damage of spinal cord injury, improves hindlimb locomotor function, and facilitates myelin regeneration. This positive effect of etanercept on spinal cord injury is probably attributable to the suppression of TNF-α, TNFR1, TNFR2, and activated caspase-3 and caspase-8 overexpressions, and the inhibition of neuronal and oligodendroglial apoptosis.

Differential regulation of TNF receptors by vagal nerve stimulation protects heart against acute ischemic injury

Vagal nerve stimulation (VS) has been reported to improve the survival after both acute and chronic myocardial infarction through the release of neurotransmitter ACh. However, the precise mechanism behind its beneficial effect is still unknown. In this study, we demonstrate the upregulation of tumor necrosis factor-alpha (TNF-α) and its cell survival TNF receptor-2 (TNFR2) as the mechanism behind VS induced myocardial protection.We investigated the effects of efferent VS on myocardial ischemic injury with in vivo and in vitro mouse models. In in vivo hearts VS significantly increased the expression of TNF-α both at the messenger and protein level after 3-hours of myocardial ischemia. In the in vitro studies ACh treatment before hypoxia, induced a significant upregulation of TNF-α compared to the untreated cardiomyocytes. Immunofluorescence analysis confirmed the synthesis of TNF-α by cardiomyocytes both in vivo and in vitro. VS also significantly reduced the myocardial infarct size (23.9±5.7% vs. 56±1.9%) and activated the cell survival Akt cascade system. Further, ACh upregulated the cell survival TNFR2 expression, while downregulating the cell destructive TNF receptor 1 (TNFR1) expression. These results were confirmed using the TNF receptors deficient mice, where the VS mediated protection was lost both in vivo and in vitro in TNFR2 (TNFR2-/-) and TNF receptors double knock out (TNFR1-/-2-/-) mice.VS and ACh protects the heart against acute ischemia or hypoxic injury by differentially regulating the TNF receptor subtypes.

Abstract 5159: Arterial Wall Expression of Tumor Necrosis Factor Receptor-2 Attenuates Atherosclerosis by Reducing Recruitment of Monocyte/Macrophages

Deficiency of tumor necrosis factor receptor-2 (TNFR2) increases TNF-induced endothelial cell apoptosis, decreases the rate of vein graft re-endothelialization, and consequently increases vein graft neointimal hyperplasia. We therefore tested the hypothesis that TNFR2 expression in the arterial wall protects against atherogenesis, by placing common carotid arterial interposition isografts from tnfr2 −/− or congenic wild type (WT) mice into the common carotid arteries of congenic apolipoprotein E −/− mice fed normal chow. We used a procedure we previously demonstrated engenders typical atherosclerosis, but in an accelerated time frame. Grafts were harvested 7 weeks post-op, perfusion-fixed, paraffin-embedded, sectioned at 4 μ m, and stained for collagen and elastin. Computerized morphometry was performed by observers blinded to specimen identity. Eccentric lesions with fibrous caps and necrotic cores developed, and demonstrated up to 85% luminal stenosis. Compared with WT carotid grafts (n=6), tnfr2 KO carotid grafts (n=5) demonstrated significantly more atherosclerosis, with 33% greater neointimal area (0.16±0.03 vs. 0.12±0.01 mm 2 , p&lt;0.01). However, medial area was equivalent in WT and tnfr2 KO arteries (0.12±0.02 vs. 0.10±0.04 mm 2 ). Outward remodeling tended to be greater in tnfr2 KO arteries (external diameter was 14±1% greater); consequently, luminal stenosis was comparable between tnfr2 KO and WT arteries (89±8% vs. 86±14%). Tnfr2 KO arteries not only developed larger neointimal lesions but also showed evidence for more unstable plaque composition: macrophage prevalence was 80±10% higher (p&lt;0.05), and SMC prevalence was 43±5% lower in tnfr2 KO than in WT arteries (p&lt;0.05), as assessed by quantitative immunofluorescence microscopy for F4/80 (macrophages) and SMC α -actin (normalized to Hoechst 33342-stained nuclear fluorescence). Concordantly, immunofluorescence of vascular cell adhesion molecule-1 per cell was 70±20% greater in tnfr2 KO carotid grafts (p&lt;0.05). We conclude that TNFR2 expression in arterial wall cells protects against atherogenesis, by mechanisms that reduce monocyte/macrophage recruitment to the atheroma.

Role of tumor necrosis factor receptor 2 (TNFR2) in colonic epithelial hyperplasia and chronic intestinal inflammation in mice

Background &Aims:Tumor necrosis factor (TNF) induces multiple effects including cell proliferation and death by ligation with TNF receptor type II (TNFR2). We studied the role of TNFR2 in chronic inflammation-induced colonic epithelial alteration. Methods: TNFR2 expression in colonic epithelial cells (CECs) was assessed by ribonuclease protection assay (RPA) and immunohistochemistry (IHC) in patients with inflammatory owel disease (IBD) and murine colitis models. TNFR2 expression was also analyzed using COL0205 cells. The role of TNFR2 in colonic epithelial homeostasis was examined by generating interleukin 6-deficient TCRαKO (αIL-6DKO) or TNFR2- deficient TCRot (otTNFR2DKO) mice. Methods:TNFR2 expression was up-regulated in CEC in both human ulcerative colitis and Crohn's disease. In vitro studies showed that TNFR2 expression was upregulated by a cooperative effect of key proinflammatory cytokines. By RPA, the increased expression of TNFR2 was detectable in TCRαK0 mice with colitis compared with TCRotKO mice without colitis or wild-type mice. In αIL-6DK0 mice, TNFR2 expression, proliferation, and nuclear factor kappa B activation of CECs were markedly reduced compared with TCRotKO mice. αTNFR2 mice also showed significantly less colonic epithelial proliferation compared with TCRotKO mice. Conclusions:Expression of TNFR2 is consistently increased on CECs in both murine colitis models as well as patients with IBD. TNFR2 may play an important role in colonic inflammation-associated alteration in the intestinal epithelium.