Presence Of Tumor Necrosis Factor Research Articles

NFκB dynamics-dependent epigenetic changes modulate inflammatory gene expression and induce cellular senescence.

Upregulation of nuclear factor κB (NFκB) signaling is a hallmark of aging and a major cause of age-related chronic inflammation. However, its effect on cellular senescence remains unclear. Here, we show that alteration of NFκB nuclear dynamics from oscillatory to sustained by depleting a negative feedback regulator of NFκB pathway, NFκB inhibitor alpha (IκBα), in the presence of tumor necrosis factor α (TNFα) promotes cellular senescence. Sustained NFκB activity enhanced inflammatory gene expression through increased NFκB-DNA binding and slowed the cell cycle. IκBα protein was decreased under replicative or oxidative stress in vitro. Furthermore, a decrease in IκBα protein and an increase in DNA-NFκB binding at the transcription start sites of age-associated genes in aged mouse hearts suggested that nuclear NFκB dynamics may play a critical role in the progression of aging. Our study suggests that nuclear NFκB dynamics-dependent epigenetic changes regulated over time in a living system, possibly through a decrease in IκBα, enhance the expression of inflammatory genes to advance the cells to a senescent state.

An oxygen-releasing agent promotes healing of skin wounds in rats.

To evaluate the local effect of a slow oxygen-release gel on the healing of standardised skin wounds caused in rats. Skin wounds were created on the backs of male rats (Rattus norvegicus, Wistar) that were randomly allocated into two groups. In the treated (T) and control (C) groups, oxygen gel and distilled water, respectively, were applied to the wounds on alternate days for 28 days. Postoperatively, euthanasia was performed at 5, 10, 14, 21 and 28 days, followed by clinical, histological (Masson's trichrome) and immunohistochemical analysis. Data were subjected to analysis of variance (ANOVA) and Bonferroni's test. The cohort comprised 50 rats. On clinical and histological analysis, groups C and T showed similar characteristics 5 days post-operation. Subsequently, group T showed better healing at 14, 21 and 28 days and presented more intense inflammatory infiltrate up to 10 days. At days 14, 21 and 28, group T exhibited a reduction in oedema and increased angiogenesis, granulation tissue formation, and deposition of collagen fibres than group C. Immunohistochemical analysis showed the presence of tumour necrosis factor (TNF)-α and vascular endothelial growth factor (VEGF) in both the groups, but the levels were significantly higher in group T (p<0.05). The local application of slow oxygen-release gel accelerated the healing of standardised skin wounds created surgically in rats, with increased angiogenesis and better collagen fibre formation.

High Expression of PR-A and Low Expression of PR-B is Correlated with Inflammation in Endometrioma Cases

BACKGROUND: Progestin therapy has been commonly used in endometriosis. The regulation of progesterone receptors B (PR-B) greatly affects the success rate of therapy in cases of endometriosis. The presence of tumor necrosis factor (TNF)-a in endometriosis triggers PR-B hypermethylation, decreasing PR-B expression and PR-B/A ratio that induce progesterone resistance. It may also occur in endometrioma. Studies regarding the distribution of PR-A and PR-B with TNF-a expression in endometriosis with endometrioma tissue samples has not been elucidated well. Therefore, this study was conducted to measure and compare the distribution of PR-A and PR-B expression, and to assess the effect of PR-B/A ratio on TNF-a in endometrioma and benign cysts.METHODS: A cross-sectional study was conducted by collecting paraffin blocks of endometriomas and benign cysts as controls, from patients undergoing surgery at Dr. Sardjito Hospital, Yogyakarta. Immunohistochemistry was performed to assess the expressions of PR-A, PR-B, TNF-a and PR-B/A ratio, to compared differences between endometriomas and benign cysts.RESULTS: Twenty-three endometrioma and 22 benign cyst tissue samples were collected. The mean PR-B expression and PR-B/A ratio were found to be lower in endometriomas than benign cysts, and mean expression of PR-A and TNF-a in endometriomas was higher than in benign cysts. However, there were no significant correlations between the expression of PR-A, PR-B, PR-B/A ratio, and TNF-a with endometriosis severity.CONCLUSION: In endometrioma cases, the expression of PR-A and TNF-a was higher, while the expression of PR-B and PR-B/A ratio was lower. However, there was no significant relationship between the ratio of PR-B/A and TNF-a.KEYWORDS: progesterone receptor, tumor necrosis factor-alpha, endometriosis, endometrioma, benign cyst, ovarian cyst

Interleukin-1 Mediates Ischemic Brain Injury via Induction of IL-17A in γδ T Cells and CXCL1 in Astrocytes

As a prototypical proinflammatory cytokine, interleukin-1 (IL-1) exacerbates the early post-stroke inflammation, whereas its neutralization is protective. To further investigate the underlying cell-type-specific IL-1 effects, we subjected IL-1 (α/β) knockout (Il1−/−) and wildtype (WT) littermate mice to permanent middle cerebral artery occlusion (pMCAO) and assessed immune cell infiltration and cytokine production in the ischemic hemisphere by flow cytometry 24 h and 72 h after stroke. Il1−/− mice showed smaller infarcts and reduced neutrophil infiltration into the ischemic brain. We identified γδ T cells and astrocytes as target cells of IL-1 signaling-mediated neutrophil recruitment. First, IL-1-induced IL-17A production in γδ T cells in vivo, and IL-17A enhanced the expression of the main neutrophil attracting chemokine CXCL1 by astrocytes in the presence of tumor necrosis factor (TNF) in vitro. Second, IL-1 itself was a potent activator of astrocytic CXCL1 production in vitro. By employing a novel FACS sorting strategy for the acute isolation of astrocytes from ischemic brains, we confirmed that IL-1 is pivotal for Cxcl1 upregulation in astrocytes in vivo. Our results underscore the pleiotropic effects of IL-1 on immune and non-immune cells within the CNS to mount and amplify the post-stroke inflammatory response.

Sitagliptin attenuates arterial calcification by downregulating oxidative stress-induced receptor for advanced glycation end products in LDLR knockout mice

Diabetes is a complex disease characterized by hyperglycemia, dyslipidemia, and insulin resistance. Plasma advanced glycation end products (AGEs) activated the receptor for advanced glycation end products (RAGE) and the activation of RAGE is implicated to be the pathogenesis of type 2 diabetic mellitus (T2DM) patient vascular complications. Sitagliptin, a dipeptidyl peptidase-4 (DPP4) inhibitor, is a new oral hypoglycemic agent for the treatment of T2DM. However, the beneficial effects on vascular calcification remain unclear. In this study, we used a high-fat diet (HFD)-fed low-density lipoprotein receptor deficiency (LDLR−/−) mice model to investigate the potential effects of sitagliptin on HFD-induced arterial calcification. Mice were randomly divided into 3 groups: (1) normal diet group, (2) HFD group and (3) HFD + sitagliptin group. After 24 weeks treatment, we collected the blood for chemistry parameters and DPP4 activity measurement, and harvested the aorta to evaluate calcification using immunohistochemistry and calcium content. To determine the effects of sitagliptin, tumor necrosis factor (TNF)-α combined with S100A12 was used to induce oxidative stress, activation of nicotinamide adenine dinucleotide phosphate (NADPH), up-regulation of bone markers and RAGE expression, and cell calcium deposition on human aortic smooth muscle cells (HASMCs). We found that sitagliptin effectively blunted the HFD-induced artery calcification and significantly lowered the levels of fasting serum glucose, triglyceride (TG), nitrotyrosine and TNF-α, decreased the calcium deposits, and reduced arterial calcification. In an in-vitro study, both S100A12 and TNF-α stimulated RAGE expression and cellular calcium deposits in HASMCs. The potency of S100A12 on HASMCs was amplified by the presence of TNF-α. Sitagliptin and Apocynin (APO), an NADPH oxidase inhibitor, inhibited the TNF-α + S100A12-induced NADPH oxidase and nuclear factor (NF)-κB activation, cellular oxidative stress, RAGE expression, osteo transcription factors expression and calcium deposition. In addition, treatment with sitagliptin, knockdown of RAGE or TNF-α receptor blunted the TNF-α + S100A12-induced RAGE expression. Our findings suggest that sitagliptin may suppress the initiation and progression of arterial calcification by inhibiting the activation of NADPH oxidase and NF-κB, followed by decreasing the expression of RAGE.

Bmi deficiency causes oxidative stress and intervertebral disc degeneration which can be alleviated by antioxidant treatment.

The transcriptional repressor Bmi‐1 is involved in cell‐cycle regulation and cell senescence, the deficiency of which has been shown to cause oxidative stress. This study investigated whether Bmi‐1 deficiency plays a role in promoting disc degeneration and the effect of treatment with antioxidant N‐acetylcysteine (NAC) on intervertebral disc degeneration. Bmi‐1−/− mice were treated with the antioxidant NAC, supplied in drinking water (Bmi‐1−/−+NAC). For in vitro experiments, mouse intervertebral discs were cultured under low oxygen tension and serum‐limiting conditions in the presence of tumour necrosis factor α and interleukin 1β in order to mimic degenerative insult. Disc metabolism parameters in these in vitro and in vivo studies were evaluated by histopathological, immunohistochemical and molecular methods. Bmi‐1−/− mice showed lower collagen Ⅱ and aggrecan levels and higher collagen Ⅹ levels than wild‐type and Bmi‐1−/−+NAC mice. Bmi‐1−/− mice showed significantly lower superoxide dismutase (SOD)‐1, SOD‐2, glutathione peroxidase (GPX)‐1 and GPX‐3 levels than their wild‐type littermates and Bmi‐1−/−+ NAC mice. Relative to Bmi‐1−/− mice, the control and Bmi‐1−/−+NAC mice showed significantly lower p16, p21, and p53 levels. These results demonstrate that Bmi‐1 plays an important role in attenuating intervertebral disc degeneration in mice by inhibiting oxidative stress and cell apoptosis.

High Polyphenol Sorghum Extract Attenuates Th2 cytokine Profile and Reduces Growth of Legionella in Raw 264.7 Mouse Macrophage Cell Line

Abstract Objectives 1) To evaluate the changes in inflammatory response induced by sorghum polyphenols in activated macrophages and 2) to evaluate possible efficacy of sorghum polyphenols on an opportunistic intracellular pathogen, Legionella pneumophila (LP). Methods Raw 265.7 cells mouse macrophage cells were treated with sorghum phenolic extract (SPE) under control and activating conditions to evaluate the role of SPE in inflammatory and anti-inflammatory processes. Study measured: nitric oxide production in the supernatant, mRNA using qPCR array of 84 genes in activated macrophages. Morphological changes were observed, and LC3 protein expression was measured to test for autophagy using western blot. NF-kB and STAT3 nuclear translocation was measured using a fractionization kit, followed by western blot. The replication of LP was measured within RAW 264.7 cells and in vitro (in media without cell presence). Cytotoxicity assay and a western blot apoptosis marker caspase-3 were used to evaluate the cytotoxicity of SPE on RAW 264.7 cells. Because LP reproduction within cells is greatly attenuated in the presence of Tumor Necrosis Factor (TNF), LP replication was measured under the presence of TNF neutralizing antibodies. Results SPE decreased nitric oxide production in activated LPS/IFNΥ macrophages although not significantly. SPE attenuated Th2 cytokine response in LPS/IFNΥ activated macrophages by decreasing expression of IL-6 and IL-10 while not changing expression of other inflammatory cytokines. Quantitative PCR data confirmed that genes in the IL-10 and IL-6 pathway were downregulated by cotreatment of SPE and LPS/IFNΥ when compared to LPS/IFNΥ alone. Morphological changes observed exhibited formation of large vacuole like structures. Analysis of LC3 confirmed that autophagy was increased in activated cells treated with SPE. Nuclear fractionization confirmed that STAT3 signaling was attenuated by SPE in activated macrophages. SPE significantly reduced the replication of LP in macrophage cells but not in vitro. The attenuation of LP grown in RAW 264.7 cells was independent of TNF presence. Conclusions This data suggests that sorghum phenolic compounds may have potential pharmaceutical/nutritional uses to combat intracellular pathogens. Funding Sources All funding was provided by the United States Department of Agriculture.

Substance P Promotes Liver Sinusoidal Endothelium-Mediated Hepatic Regeneration by NO/HGF Regulation.

Liver sinusoidal endothelial cells (LSECs) are highly specialized and involved in hepatic regeneration by interacting with hepatic stellate cells (HSCs) and hepatocytes in a paracrine manner. However, hepatic injury can impair cellular activity and lead to endothelial dysfunction, eventually inducing the development of critical hepatic disease, including cirrhosis. Because LSECs exert their effects through paracrine factors, maintenance of paracrine potentials and survival activity in LSECs under injury stress is a critical strategy for inhibiting disease progression. This study explored the effect of Substance-P (SP) on cell viability, proliferation, and nitric oxide (NO)/hepatocyte growth factor (HGF) production in LSECs. Under noninjured conditions, SP treatment enhanced cell viability, cell proliferation, and HGF/NO secretion in LSECs. In the presence of tumor necrosis factor (TNF)-α-induced inflammatory stress, SP blocked TNF-α-induced endothelial dysfunction, accompanied by elevated cell viability and NO/HGF secretion. Interestingly, SP-primed LSEC-conditioned medium accelerated hepatocyte repopulation without causing morphological alterations. The primitive effect of SP was reversed by endothelial nitric oxide synthase inhibitor or HGF/c-MET inhibitor, indicating the importance of the NO/HGF combination in hepatic regeneration by SP. Taken together, these results suggest SP can protect LSECs from inflammatory stress by NO/HGF, which contributes to hepatocyte repopulation.

Milk-Derived Tripeptides IPP (Ile-Pro-Pro) and VPP (Val-Pro-Pro) Enhance Insulin Sensitivity and Prevent Insulin Resistance in 3T3-F442A Preadipocytes.

There is great interest in developing naturally derived compounds, especially bioactive peptides with potential insulin sensitizing effects and/or preventing insulin resistance. Previously, we showed adipogenic and insulin mimetic actions of IPP (Ile-Pro-Pro) and VPP (Val-Pro-Pro), the milk-derived tripeptides on cultured preadipocytes, in addition to their previously characterized antihypertensive and anti-inflammatory functions. However, the effect of these peptides on insulin signaling is not known. Therefore, we examined IPP and VPP effects on insulin signaling in preadipocytes, a well-established model for studying insulin signaling. Our results suggested both peptides enhanced insulin signaling and contributed toward the prevention of insulin resistance in the presence of tumor necrosis factor (TNF). Inhibition of inflammatory mediator NF-kB under TNF stimulation was a likely contributor to the prevention of insulin resistance. VPP further enhanced the expression of glucose transporter 4 (GLUT4) in adipocytes and restored glucose uptake in TNF-treated adipocytes. Our data suggested the potential of these peptides in the management of conditions associated with impairments in insulin signaling.

Anticancer effect of Dracaena arborea leaf extract through down-regulation of VCAM-1 and EMT proteins via suppressing Akt/PKC pathway

Dracaena arborea (Willd.) Link is known to be used in traditional medicine for the treatment of various dis-eases. Because it possesses steroidal saponins and flavonoids which display a variety of biological actions including anti-car-cinogenic, it might exhibit anti-cancer effect. However, the anti-cancer effect of D. arborea is not well known. Therefore, in this study, we investigated anti-cancer effect of D. arborea on highly metastatic human breast cancer cells MDA-MB-231. Methanolic extract of leaves of Dracaena arborea (MEDA) decreased cell viability of MDA-MB-231 cells, but not endothelial cells (ECs), in a dose-dependent manner for 24 h. Then, pretreatment of MEDA significantly inhibited the binding of MDA-MB-231 to ECs and the invasion of MDA-MB-231 in the presence of tumor necrosis factor (TNF)-α or not. Pretreatment of MEDA significantly inhibited vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) expression both in TNF-α-treated MDA-MB-231 cells and ECs at the indicated doses, and the inhibitory effect was more prominent in VCAM-1 rather than ICAM-1. TNF-α-mediated induction of β-catenin and Snail-1 and secretion of matrix metalloproteinase-9 (MMP-9) were significantly down-regulated by MEDA from 50 μg/ml and 100 μg/ml, respectively. Fur-thermore, MEDA effectively down-regulated protein kinase C (PKC) and Akt activation by TNF-α, suggesting that inhi-bition of PKC and Akt pathways by MEDA are responsible for differential inhibition of VCAM-1 and the epithelial&#8211; mesenchymal transition (EMT)-related proteins β-catenin and Snail-1. Taken together, MEDA exhibits anti-cancer effects through inhibition of adhesion of cancer cells to ECs and the invasion of cancer cells through down-regulation of VCAM-1 and EMT proteins via suppressing Akt/PKC pathway, suggesting a possibility to be served as a therapeutic agent against cancer metastasis.

Crataegus Special Extract WS 1442 Effects on eNOS and microRNA 155.

Increased expression of microRNA 155 (miR-155) results in a decrease in endothelial nitric oxide synthase (eNOS) expression and impaired endothelial function. Factors that have been shown to increase expression of miR-155 may be mitigated by WS 1442, an extract of hawthorn leaves and flowers (Crataegus special extract) that contains a range of pharmacologically active substances including oligomeric proanthocyanidins and flavonoids. The purpose of this study is to determine the effect of WS 1442 on the expression of miR-155 and eNOS in the presence of tumor necrosis factor (TNF-α). Human umbilical vein endothelial cells (HUVECs) were studied after the exposure to TNF-α, with or without simvastatin (positive control) and WS 1442. The expression levels of eNOS, phosphorylated eNOS, and miR-155 in the different HUVEC treatment groups were determined by western blot and quantitative real-time polymerase chain reaction, respectively. To evaluate the effect of WS 1442 on the eNOS activity, the medium and intracellular nitrate/nitrite (NO) concentrations were also analyzed using a colorimetric Griess assay kit. The results demonstrated that TNF-α upregulated miR-155 expression and decreased eNOS expression and NO concentrations. WS 1442 also increased miR-155 expression and decreased eNOS expression but, unlike TNF-α, increased phosphorylated eNOS expression and NO concentrations. Surprisingly, WS 1442 increased miR-155 expression; however, WS 1442 mitigated the overall negative effect of miR-155 on decreasing eNOS expression by increasing expression of phosphorylated eNOS and resulting in an increase in NO concentrations. In the setting where miR-155 may be expressed, WS 1442 may offer vascular protection by increasing the expression of phosphorylated eNOS.

P098 Aryl hydrocarbon receptor ameliorates intestinal barrier dysfunction in colitis by regulating the myosin light-chain kinase signalling pathway

Accumulating evidence suggests that the aryl hydrocarbon receptor (AhR) plays an important role in regulating the function of the intestinal barrier, especially the immune response, in patients with inflammatory bowel disease and in mouse models. However, whether AhR aids to protect the intestinal mucosal mechanical barrier remains unknown. In this study, we hypothesised that AhR ameliorates intestinal mucosal mechanical barrier in colitis. Experimental colitis was induced in male C57BL/6 wild-type mice and AhR knockout (AhR KO) mice using 2,4,6-trinitrobenzenesulfonic acid (TNBS). Correspondingly, the AhR agonist 6-formylindolo [3,2-b] carbazole (FICZ) and the AhR antagonist CH223191 were used to treat Caco-2 monolayers in the presence of tumour necrosis factor α (TNF-α). Colonic mucosal barrier permeability and myosin light-chain kinase (MLCK) signalling pathway were investigated both in vivo and vitro. In vivo experiments, it was found that TNBS-induced colitis was more severe in AhR KO mice than in C57BL/6 wild-type mice. The intestinal permeability was significantly higher in AhR KO mice than in wild-type mice induced by TNBS. The expression and fluorescence of tight junction protein in AhR KO mice were lower than that in wild-type mice induced by TNBS. The increased expression of MLCK and phosphorylated MLC (pMLC) indicated that this pathway was open. In cell culture experiments, treatment with FICZ caused retention of the tight junction protein, alleviated the increase of intestinal permeability, and mitigated epithelial injury. However, treatment with CH223191 facilitated the unblocking of the MLCK-pMLC signalling pathway and repressed the protein expression of tight junction in vitro. AhR plays an important role in ameliorating epithelial barrier dysfunction in colitis through regulating the MLCK signalling pathway, suggesting that AhR may be a suitable entrance of addressing this condition.

Osteoclast Differentiation is Downregulated by Select Polyphenolic Fractions from Dried Plum via Suppression of MAPKs and Nfatc1 in Mouse C57BL/6 Primary Bone Marrow Cells

BackgroundClinical and preclinical studies have shown that dietary supplementation with dried plum improves bone health. These osteoprotective effects are a result, in part, of the antiresorptive properties of the fruit, which appear to be mediated by its polyphenolic compounds. ObjectiveThis study was designed to determine if certain fractions of the polyphenolic compounds in dried plums are responsible for the antiresorptive effects and whether they alter mitogen-activated protein kinase (MAPK) and calcium signaling, which are essential to osteoclast differentiation and activity, under normal and inflammatory conditions. MethodsSix polyphenolic fractions were derived from the total polyphenolic extract of dried plum based on solubility. Initial screening, with the use of the Raw 264.7 monocyte and macrophage cell line, showed that 3 fractions had the most marked capacity to downregulate osteoclast differentiation. This response was confirmed in 2 of the fractions by using primary bone marrow–derived cultures and in all subsequent experiments to determine how osteoclast differentiation and function were altered with a focus on these 2 fractions in primary cultures. Data were analyzed by using ANOVA followed by post hoc analyses. ResultsBoth of the polyphenol fractions decreased osteoclast differentiation and activity coincident with downregulating nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 1 (Nfatc1), which is required for osteoclast differentiation. Calcium signaling, essential for the auto-amplification of Nfatc1, was suppressed by the polyphenolic fractions under normal conditions as indicated by suppressed mRNA expression of costimulatory receptors osteoclast-associated receptor (Oscar), signaling regulatory protein β1 (Sirpb1), and triggering receptor expressed on myeloid cells 2 (Trem2). In contrast, in the presence of tumor necrosis factor α (TNF-α), only Sirpb1 was downregulated. In addition to calcium signaling, phosphorylation of extracellular signal–regulated kinase (Erk) and p38 MAPK, involved in the expression and activation of Nfatc1, was also suppressed by the polyphenolic fractions. ConclusionThese results show that certain types of polyphenolic compounds from dried plum downregulate calcium and MAPK signaling, resulting in suppression of Nfatc1 expression, which ultimately decreases osteoclast formation and activity.

Abstract 962: Novel targeting of tumor-infiltrating TNFR2 Tregs: removing suppression with microenvironment-specific antibodies

Abstract Background: A recognized and potent subtype of regulatory T cells (Tregs) expresses tumor necrosis factor receptor 2 (TNFR2), a member of the tumor necrosis factor (TNF) superfamily. TNFR2 expressing Tregs are overabundant in human and murine tumors and are the most potent suppressors of host immune responses. In addition, recent data in diverse human tumors show that TNFR2-expressing host T cells are the dominant Tregs in the tumor microenvironment, expressed far in excess of OX40 and GITR. Unlike many tumor markers, TNFR2 has limited expression in the immune system and almost no expression on parenchymal cells. This makes TNFR2 Tregs an attractive target for anticancer therapy. Methods: We produced monoclonal antibodies to human TNFR2 with the goal of inactivating the potent host Tregs that express TNFR2, competing with the agonism of TNF and inactivating infiltating Tregs of human tumors. Tregs from fresh ovarian ascites fluid were compared to Tregs from normal human blood donors for antibody potency. Results: Using a 48-hr cell-based Treg assay, we showed that TNFR2 antagonistic antibodies can inhibit Treg proliferation, suppress soluble TNFR2 secretion on normal cells and allow T effector expansion. The antagonism is dominant, succeeding even in the presence of TNF, which is a TNFR2 agonist. Furthermore, the structural biology of dominant TNFR2 receptor antagonism uncovers a unique conformation that equally inhibits downstream NFkB signaling. We also showed that dominant TNFR2 antibodies in a dose dependent fashion kill Tregs in ovarian cancer infiltrates more strongly than Tregs from healthy donors. Treg killing in the cancer microenvironment was dependent on cell proliferation and turnover the Tregs. Conclusion: In sum, blocking TNFR2 signaling on the most potent Tregs of the tumor microenvironment with a dominant antagonist antibody selectively heightens death of the immunosuppressive Tregs in the tumor microenvironment, at least in ovarian cancer. With the expanding knowledge that TNFR2 Tregs are the most potent host cells of the immune response, their dominance in diverse human tumor infiltrates and the natural low expression of this marker on normal lymphoid cells makes targeting of TNFR2 in the tumor microenvironment attractive. Citation Format: Heather Torrey, John Butterworth, Toshiyuki Mera, Yoshiaki Okubo, Limei Wang, Danielle Baum, Audrey Defusco, Sara Plager, Sarah Warden, Daniel Huang, Eva Vanamee, Rosemary Foster, Denise L. Faustman. Novel targeting of tumor-infiltrating TNFR2 Tregs: removing suppression with microenvironment-specific antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 962. doi:10.1158/1538-7445.AM2017-962

TNF Signaling in Peritoneal Mesothelial Cells: Pivotal Role of cFLIPL.

♦ BACKGROUND: Peritoneal dialysis (PD) coincides with high concentrations of proinflammatory cytokines, such as tumor necrosis factor (TNF), in the peritoneal cavity. During treatment, chronic inflammatory processes lead to damage of the peritoneal membrane and a subsequent ultrafiltration failure. Human peritoneal mesothelial cells (HPMCs) play a central role as mediators and targets of PD-related inflammatory changes. Although TNF Receptor 1 (TNFR1) is expressed in high numbers on the cells, TNF-induced apoptosis is inhibited. Here, the underlying molecular mechanisms of TNFR1 signaling in HPMCs are investigated. ♦ METHODS: Human peritoneal mesothelial cells were isolated from the omentum of healthy donors and the dialysis solution of PD patients. Flow cytometry was applied to determine cell surface expression of TNFR1 on HPMCS from healthy donors in absence or presence of TNF or PD fluid (PDF) and were compared to TNFR1 expression on cells from PD patients. To investigate TNFR1-mediated signaling, HPMCs were treated with PDF or TNF, and expression patterns of proteins involved in the TNFR1 signaling pathway were assessed by western blot. ♦ RESULTS: Incubation with PDF led to a significant up-regulation of TNFR1 on the cell surface correlating with elevated TNFR1 numbers on HPMCs from PD patients. Investigations of underlying molecular mechanisms of TNFR1 signaling showed that PDF affects TNFR1 signaling at the proapoptotic signaling pathway by upregulation of IκBα and downregulation of cFLIPL. In contrast, TNF exclusively induces the activation of NFκB by an increase of phosphorylated IκBα. ♦ CONCLUSIONS: Novel and relevant insights into the mechanisms of TNFR1-mediated signaling in HPMCs with an impact on our understanding of PD-associated damage of the peritoneal membrane are shown.

Simple in vitro generation of human leukocyte antigen-G–expressing T-regulatory cells through pharmacological hypomethylation for adoptive cellular immunotherapy against graft-versus-host disease

BackgroundMajor barriers in using classical FOXP3+ regulatory T cells (Tregs) in clinical practice are their low numbers in the circulation, the lack of specific cell surface markers for efficient purification and the loss of expression of Treg signature molecules and suppressive function after in vitro expansion or in a pro-inflammatory microenviroment. A surface molecule with potent immunosuppressive function is the human leukocyte antigen-G (HLA-G), which is normally expressed in placenta protecting the “semi-allogeneic” fetus from maternal immune attack. Because HLA-G expression is strongly regulated by methylation, we asked whether hypomethylating agents (HA) may be used in vitro to induce HLA-G expression on conventional T cells and convert them to Tregs. MethodsHuman peripheral blood T cells were exposed to azacytidine/decitabine and analyzed for HLA-G expression and their in vitro suppressor properties. ResultsHA treatment induces de novo expression of HLA-G on T cells through hypomethylation of the HLA-G proximal promoter. The HA-induced CD4+HLA-Gpos T cells are FOXP3 negative and have potent in vitro suppression function, which is dependent to a large extent, but not exclusively, on the HLA-G molecule. Converted HLA-Gpos suppressors retain their suppressor function in the presence of tumor necrosis factor (TNF) and preserve hypomethylated the HLA-G promoter for at least 2 days after azacytidine exposure. Decitabine-treated T cells suppressed ex vivo the proliferation of T cells isolated from patients suffering from graft-versus-host disease (GVHD). DiscussionWe propose, in vitro generation of HLA-G–expressing T cells through pharmacological hypomethylation as a simple, Good Manufacturing Practice (GMP)-compatible and efficient strategy to produce a stable Treg subset of a defined phenotype that can be easily purified for adoptive immunotherapy.

Vibrio cholerae Cholix Toxin-Induced HepG2 Cell Death is Enhanced by Tumor Necrosis Factor-Alpha Through ROS and Intracellular Signal-Regulated Kinases.

Cholix toxin (Cholix) from Vibrio cholerae is a potent virulence factor exhibiting ADP-ribosyltransferase activity on eukaryotic elongation factor 2 (eEF2) of host cells, resulting in the inhibition of protein synthesis. Administration of Cholix or its homologue Pseudomonas exotoxin A (PEA) to mice causes lethal hepatocyte damage. In this study, we demonstrate cytotoxicity of Cholix on human hepatocytes in the presence of tumor necrosis factor α (TNF-α), which has been reported to play a fatal role in PEA administered to mice. Compared with incubating HepG2 cells with Cholix alone, co-treatment with TNF-α and Cholix (TNF-α/Cholix) significantly enhanced the activation of caspases, cytochrome c release from mitochondria into cytoplasm, and poly-ADP-ribose polymerase (PARP) cleavage, while incubation with TNF-α alone or co-treatment with TNF-α/catalytically inactive Cholix did not. In the early stage of cell death, Cholix increased phosphorylation of mitogen-activated protein kinases (e.g., p38, ERK, JNK) and Akt, which was not affected by TNF-α alone. MAPK inhibitors (SP600125, SB20852, and U0126) suppressed PARP cleavage induced by TNF-α/Cholix. Protein kinase inhibitor Go6976 suppressed JNK phosphorylation and PARP cleavage by TNF-α/Cholix. In contrast, PKC activator PMA in the absence of TNF-α promoted Cholix-induced PARP cleavage. Reactive oxygen species (ROS) inhibitor, N-acetyl cysteine (NAC), suppressed TNF-α/Cholix-induced JNK and ERK phosphorylation, resulting in inhibition of PARP cleavage. These data suggest that ROS and JNK pathways are important mediators of TNF-α/Cholix-induced HepG2 cell death.

Regulation of cyclic AMP response element-binding protein during neuroglial interactions.

Communications between neurons and glial cells play an important role in regulating homeostasis in the central nervous system. cAMP response element-binding protein (CREB), a transcription factor, is down-regulated by neurotoxins, which are known to be released by activated glial cells. To determine the role of CREB signaling in neuroglial interactions, we used three neuroglial coculture models consisting of human neuroprogenitor cell (NPC)-derived neurons and human microglia. Conditioned medium from the Abeta (Aβ)-activated microglia decreased CREB phosphorylation and brain-derived neurotrophic factor promoter activity (47%), whereas the same medium induced (p < 0.01) the promoter of CXCL10, a chemokine, in NPC-derived neuron-rich cultures. These effects were reversed when microglia were exposed to Aβ in the presence of minocycline, an anti-inflammatory agent. The expression of CREB targets, including brain-derived neurotrophic factor, synapsin-1, and BIRC3 decreased by 50-65% (p < 0.01) in neurons isolated by laser capture microdissection in close proximity of microglia in neuroglial mixed cultures. Neuronal survival actively modulated microglial behavior when neurons and microglia were cocultured side-by-side on semicircles of ACLAR membrane. Neuronal injury, caused by the over-expression of dominant negative form of CREB, exacerbated Aβ-mediated microglial activation, whereas CREB over-expression resulted in decreased microglial activation. Decreases in the levels of neuronal markers were observed when NPCs were differentiated in the presence of proinflammatory cytokines IL-1β, tumor necrosis factor α, or IL-6. Instead, the NPCs differentiated into a glial phenotype, and these effects were more pronounced in the presence of tumor necrosis factor α. Our findings suggest that CREB down-regulation is an important component of defective neuroglial communications in the brain during neuroinflammation. Neuroglial interactions were examined using coculture models of human neuroprogenitor cell-derived neurons and microglia isolated from human fetal brain. A novel coculture model of neurons and microglia cultured on ACLAR membranes in the same dish was also included. In this model, over-expression of the dominant negative mutant form of the transcription factor CREB in neurons induced neuronal apoptosis and microglial activation whereas expression of the wild type form of CREB resulted in protection of neurons and suppressed microglial activity, thereby suggesting that neurons play an active role in neuroglial interactions.

Selective inhibition of TNFR1 reduces osteoclast numbers and is differentiated from anti-TNF in a LPS-driven model of inflammatory bone loss

The treatment of autoimmune disorders has been revolutionised by the introduction of biologics such as anti-tumour necrosis factor (anti-TNF). Although in rheumatoid arthritis patients a bone sparing effect of anti-TNF has been shown, the mechanism is not fully understood. Anti-TNF molecules block tumour necrosis factor (TNF) and prevent signalling via both TNF receptor 1 (TNFR1; p55) and TNF receptor 2 (TNFR2; p75). However, signalling via TNFR2 is reported to have protective effects in a number of cell and organ systems. Hence we set out to investigate if pharmacological inhibition of TNFR1 had differential effects compared to pan-TNF inhibition in both an in vitro cell-based model of human osteoclast activity and an in vivo mouse model of lipopolysaccharide (LPS)-induced osteolysis. For the in vitro experiments the anti-human TNFR1 domain antibody (dAb) DMS5541 was used, whereas for the in vivo mouse experiments the anti-mouse TNFR1 dAb DMS5540 was used. We show that selective blocking of TNFR1 signalling reduced osteoclast formation in the presence of TNF. Subcutaneous LPS injection over the calvaria leads to the development of osteolytic lesions within days due to inflammation driven osteoclast formation. In this model, murine TNFR2 genetically fused with mouse IgG1 Fc domain (mTNFR2.Fc), an anti-TNF, did not protect from bone loss in contrast to anti-TNFR1, which significantly reduced lesion development, inflammatory infiltrate, and osteoclast number and size. These results support further exploring the use of TNFR1-selective inhibition in inflammatory bone loss disorders such as osteomyelitis and peri-prosthetic aseptic loosening.

The novel S59P mutation in the TNFRSF1A gene identified in an adult onset TNF receptor associated periodic syndrome (TRAPS) constitutively activates NF-κB pathway

IntroductionMutations in the TNFRSF1A gene, encoding tumor necrosis factor receptor 1 (TNF-R1), are associated with the autosomal dominant autoinflammatory disorder, called TNF receptor associated periodic syndrome (TRAPS). TRAPS is clinically characterized by recurrent episodes of long-lasting fever and systemic inflammation. A novel mutation (c.262 T > C; S59P) in the TNFRSF1A gene at residue 88 of the mature protein was recently identified in our laboratory in an adult TRAPS patient. The aim of this study was to functionally characterize this novel TNFRSF1A mutation evaluating its effects on the TNF-R1-associated signaling pathways, firstly NF-κB, under particular conditions and comparing the results with suitable control mutations.MethodsHEK-293 cell line was transfected with pCMV6-AC construct expressing wild-type (WT) or c.262 T > C (S59P), c.362G > A (R92Q), c.236C > T (T50M) TNFRSF1A mutants. Peripheral blood mononuclear cells (PBMCs) were instead isolated from two TRAPS patients carrying S59P and R92Q mutations and from five healthy subjects. Both transfected HEK-293 and PBMCs were stimulated with tumor necrosis factor (TNF) or interleukin 1β (IL-1β) to evaluate the expression of TNF-R1, the activation of TNF-R1-associated downstream pathways and the pro-inflammatory cytokines by means of immunofluorescent assay, array-based technique, immunoblotting and immunometric assay, respectively.ResultsTNF induced cytoplasmic accumulation of TNF-R1 in all mutant cells. Furthermore, all mutants presented a particular set of active TNF-R1 downstream pathways. S59P constitutively activated IL-1β, MAPK and SRC/JAK/STAT3 pathways and inhibited apoptosis. Also, NF-κB pathway involvement was demonstrated in vitro by the enhancement of p-IκB-α and p65 nuclear subunit of NF-κB expression in all mutants in the presence of TNF or IL-1β stimulation. These in vitro results correlated with patients’ data from PBMCs. Concerning the pro-inflammatory cytokines secretion, mainly IL-1β induced a significant and persistent enhancement of IL-6 and IL-8 in PBMCs carrying the S59P mutation.ConclusionsThe novel S59P mutation leads to defective cellular trafficking and to constitutive activation of TNF-R1. This mutation also determines constitutive activation of the IL-1R pathway, inhibition of apoptosis and enhanced and persistent NF-κB activation and cytokine secretion in response to IL-1β stimulation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0604-7) contains supplementary material, which is available to authorized users.