Mechanism Of Tumor Necrosis Factor Research Articles

The mechanisms of tumor necrosis factor α in regulating Krüpple-like factor 4 expression in SK-BR-3 breast cancer cells.

To explore the expression and functional role of Krüpple-like factor 4 (KLF4) protein stimulated by tumor necrosis factor α (TNF-α) in SK-BR-3 breast cancer cells. SK-BR-3 cells were stimulated with various concentrations of TNF-α at 0, 1, 5, 10, and 20ng/mL. Expression levels of KLF4 protein were detected by Western blotting. In the detection of apoptosis, flow cytometry, and DAPI staining were used for detecting the level of apoptosis. KLF4 expression was markedly elevated following stimulation of SK-BR-3 with TNF-α. At the same time, the expression of KLF4 protein increased gradually with the increase of TNF-α stimulation concentration. TNF-α stimulation of SK-BR-3 cells increased apoptosis as measured by apoptosis levels. By overexpressing KLF4 protein in SK-BR-3 cells, it similarly increased apoptosis and promoted cell death of SK-BR-3 cells. TNF-α promotes KLF4 expression, while TNF-α promotes apoptosis in SK-BR-3 cells, a process that may be due to elevated KLF4 protein expression.

Effect and mechanism of TNF-α and etanercept on the invasion ability of extravillous trophoblast cell in URSA patients

Objective: To investigate the effect and mechanism of tumor necrosis factor α (TNF-α) and its inhibitor etanercept (ETA) on the invasion ability of extravillous trophoblast in patients with unexplained recurrent spontaneous abortion (URSA). Methods: (1) Patients were collected from March to June in 2019. They were divided into the URSA group (n=15) and the normal control group (n=15), according to whether diagnosed with URSA or not. The mRNA expression levels of TNF-α in villi tissue of patients in the two groups were detected by quantitative real-time PCR (qRT-PCR). (2) The mRNA and protein expression levels of matrix metalloproteinase-2 (MMP-2), Slug and CXC chemokine rceptor 4 (CXCR4) in HTR-8/SVneo cells were detected by qRT-PCR or western blot after being stimulated by exogenous TNF-α (0.2, 2, 20 ng/ml) alone or TNF-α along with ETA, or phosphate buffered saline (PBS) as control. (3) The invasion ability of HTR-8/SVneo cells was investigated by transwell test after stimulating by TNF-α alone or TNF-α along with ETA. (4) The mRNA and protein expression levels of MMP-2, Slug and CXCR4 in HTR-8/SVneo cells, which were stimulated by TNF-α (2 ng/ml) alone after nuclear factor-κB (NF-κB) inhibitor, BAY 11-7028, preconditioning, were detected by qRT-PCR or western blot. Results: (1) The mRNA expression level of TNF-α in villi tissue of URSA group (4.10±0.49) was 4.1 times as much as the normal control group (t=10.51, P<0.05). (2) The mRNA and protein expression levels of MMP-2, Slug and CXCR4 in HTR-8/SVneo cells of TNF-α group were significantly lower than those in PBS control group (P<0.05) and those in TNF-α along with ETA group (P<0.05). (3) The invasion ability of HTR-8/SVneo cells in TNF-α group was significantly decreased than PBS group and TNF-α along with ETA group (78±14 vs 373±26 vs 227±44, P<0.05). (4) The mRNA and protein expression levels of MMP-2, Slug and CXCR4 in HTR-8/SVneo cells with BAY 11-7028 preconditioning (mRNA: 1.03±0.10, 1.03±0.06, 1.09±0.08; protein: 1.09±0.03, 1.49±0.03, 1.12±0.03) were significantly higher than without preconditioning after being stimulated by TNF-α (all P<0.05). Conclusions: The expression of TNF-α in the villi of URSA patients is much higher than normal early pregnant women. TNF-α could decrease the capacity of invasion by suppressing the expression of MMP-2, Slug and CXCR4 through NF-κB signaling pathway in extravillous trophoblast cells. While ETA could improve the invasiveness capability of extravillous trophoblast cells through inhibiting the negative effect of TNF-α.

Blocking tumor necrosis factor-α delays progression of chronic obstructive pulmonary disease in rats through inhibiting MAPK signaling pathway and activating SOCS3/TRAF1.

The present study was conducted in order to study the detailed molecular mechanism of tumor necrosis factor (TNF)-α in chronic obstructive pulmonary disease (COPD). The rats were treated with cigarette smoke (CS) and lipopolysaccharide (LPS) to establish the COPD model. Next, the changes in lung injury in COPD rats with TNF-α knockdown was tested. Meanwhile, the regulation of TNF-α on MAPK pathway and its downstream molecules (SOCS3/TRAF1) was determined by western blotting. On this basis, the activation of MAPK and inhibition of SOCS3/TRAF1 was also examined. Subsequently, the lung function was tested with the plethysmograph, the cells of bronchoalveolar lavage fluid was counted and classified. Furthermore, lung tissue sections were stained with hematoxylin and eosin to verify whether the treatment of MAPK pathway and downstream molecules affected the effect of TNF-α knockdown on COPD. The present study showed that TNF-α knockdown could alleviate the decrease in the function and inflammatory injury of the lungs of rats with COPD. Western blot analysis verified that TNF-α knockdown could inhibit the activation of MAPK pathway and increase the expression of SOCS3/TRAF1. The following experimental results showed that the relief of lung injury caused by TNF-α knockdown could be deteriorated by activating MAPK pathway. It was also found that the symptom of COPD was decreased following transfection with sh-TNF-α but worsened by SOCS3/TRAF1 knockdown. Overall, TNF-α knockdown inhibited the activation of MAPK pathway and increased the expression of SOCS3/TRAF1, thus delaying the process of COPD.

Potential Molecular Mechanism of TNF Superfamily-Related Genes in Glioblastoma Multiforme Based on Transcriptome and Epigenome.

Objective: This study aimed to investigate the molecular mechanism of tumor necrosis factor (TNF) superfamily-related genes and potential therapeutic drugs for glioblastoma multiforme (GBM) patients based on transcriptome and epigenome.Methods: Gene expression data, corresponding clinical data, and methylation data of GBM samples and normal samples in the TCGA-GBM and GTEx datasets were downloaded. The TNF-related genes were obtained, respectively, from two groups in the TCGA dataset. Then, the TNF-related differentially expressed genes (DEGs) were investigated between two groups, followed by enrichment analysis. Moreover, TNF superfamily-related gene expression and upstream methylation regulation were investigated to explore candidate genes and the prognostic model. Finally, the protein expression level of candidate genes was performed, followed by drug prediction analysis.Results: A total of 41 DEGs including 4 ligands, 18 receptors, and 19 downstream signaling molecules were revealed between two groups. These DEGs were mainly enriched in pathways like TNF signaling and functions like response to TNF. A total of 5 methylation site-regulated prognosis-related genes including TNF Receptor Superfamily Member (TNFRSF) 12A, TNFRSF11B, and CD40 were explored. The prognosis model constructed by 5 genes showed a well-prediction effect on the current dataset and verification dataset. Finally, drug prediction analysis showed that zoledronic acid (ZA)-TNFRSF11B was the unique drug–gene relation in both two databases.Conclusion: Methylation-driven gene TNFRSF12A might participate in the development of GBM via response to the TNF biological process and TNF signaling pathway and significantly associated with prognosis. ZA that targets TNFRSF11B expression might be a potential effective drug for clinical treatment of GBM.

Tumour necrosis factor-α promotes BMHSC differentiation by increasing P2X7 receptor in oestrogen-deficient osteoporosis.

The exact mechanism of tumour necrosis factor α (TNF‐α) promoting osteoclast differentiation is not completely clear. A variety of P2 purine receptor subtypes have been confirmed to be widely involved in bone metabolism. Thus, the purpose of this study was to explore whether P2 receptor is involved in the differentiation of osteoclasts. Mouse bone marrow haematopoietic stem cells (BMHSCs) were co‐cultured with TNF‐α to explore the effect of TNF‐α on osteoclast differentiation and bone resorption capacity in vitro, and changes in the P2 receptor were detected at the same time. The P2 receptor was silenced and overexpressed to explore the effect on differentiation of BMHSCs into osteoclasts. In an in vivo experiment, the animal model of PMOP was established in ovariectomized mice, and anti‐TNF‐α intervention was used to detect the ability of BMHCs to differentiate into osteoclasts as well as the expression of the P2 receptor. It was confirmed in vitro that TNF‐α at a concentration of 20 ng/mL up‐regulated the P2X7 receptor of BMHSCs through the PI3k/Akt signalling pathway, promoted BMHSCs to differentiate into a large number of osteoclasts and enhanced bone resorption. In vivo experiments showed that more P2X7 receptor positive osteoclasts were produced in postmenopausal osteoporotic mice. Anti‐TNF‐α could significantly delay the progression of PMOP by inhibiting the production of osteoclasts. Overall, our results revealed a novel function of the P2X7 receptor and suggested that suppressing the P2X7 receptor may be an effective strategy to delay bone formation in oestrogen deficiency‐induced osteoporosis.

Effects and mechanism of exogenous tumor necrosis factor α on differentiation of mesenchymal stem cells of mice into sweat gland cells in three-dimensional environment

Objective: To explore the effects and molecular mechanism of tumor necrosis factor α (TNF-α) on differentiation of mesenchymal stem cells of mice into sweat gland cells in a three-dimensional environment. Methods: (1) Five 6-8 week-old female C57BL/6 mice were used, with one 1 cm(2) deep partial-thickness to full-thickness scald wound being created on the back of each mouse with a scald apparatus. One day after injury, the full-thickness skin tissue of the wound was taken, and the concentration of TNF-α in the tissue was detected by enzyme-linked immunosorbent assay. (2) Gelatin in the mass of 0.9 g and 0.3 g sodium alginate were mixed and then dissolved in 30 mL phosphate buffer solution to make hydrogel. Full-thickness skin of the planta of 10 male and female one day newborn C57BL/6 mice was ground into dermal homogenate. The mesenchymal stem cells were isolated from femur and tibia of 10 male and female C57BL/6 mice born for 7 days and cultured. A final density of 1.5×10(5) cells/mL of bioink was made of mixture of 8 mL pre-warmed hydrogel, 1 mL mouse foot dermal homogenate, and 1 mL the second or third passage of mesenchymal stem cell suspensions. The three-dimensional bioprinter was used to print 12 cylindrical blocks arranged in a crisscross pattern in petri dish. The printed blocks were cross-linked with 25 g/L calcium chloride solution for 10 min and then cultured for 12 hours by adding a medium for mesenchymal stem cells. Subsequently, the printed blocks were divided into blank control group and TNF-α treatment group according to the random number table, with 6 plates and 6 blocks in each group. Both groups of printed blocks were cultured with fresh sweat gland induction medium, and a final mass concentration of 20 ng/mL TNF-α was added into the medium of TNF-α treatment group. After 6 hours of culture, the mRNA expression of pluripotency marker Nanog in the mesenchymal stem cells of two plates of each group was detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR), and the protein expression of Nanog in the mesenchymal stem cells of one plate of each group was detected by Western blotting, both with triplicate samples. After 14 days of culture, the mRNA expression of sweat gland cell markers cytokeratin 14 (CK14), CK18, sodium potassium adenosine triphosphatase protein a1 (ATP1a1), and aquaporin 5 (AQP5) was detected by real-time fluorescent quantitative RT-PCR in the mesenchymal stem cells of 2 plates of each group (n=3), and the protein expression distribution of CK14, CK18, ATP1a1, and AQP5 of the mesenchymal stem cells in one plate of each group was detected by immunofluorescence staining. Data were statistically analyzed with independent sample t test. Results: (1) One day after injury, the mass concentration of TNF-α in the scald wound tissue of mouse was (19±3) ng/mL. (2) After 6 hours of culture, the mRNA and protein expression levels of Nanog in the mesenchymal stem cells of printed blocks in TNF-α treatment group were 0.39±0.04 and 0.36±0.03, respectively, which were significantly lower than 1.00±0.05 and 1.00±0.07 of blank control group (t=16.51, 14.56, P<0.01). (3) After 14 days of culture, the mRNA expression levels of CK18, CK14, ATP1a1, and AQP5 in the mesenchymal stem cells of printed blocks in TNF-α treatment group were 0.38±0.03, 0.42±0.11, 0.23±0.06, and 0.25±0.03, respectively, which were significantly less than 1.00±0.03, 1.00±0.05, 1.00±0.05, 1.00±0.07 of blank control group (t=25.31, 8.31, 17.07, 17.06, P<0.01). (4) After 14 days of culture, the CK18, CK14, ATP1a1, and AQP5 protein were widely distributed in the cytoplasm of mesenchymal stem cells in printed blocks of blank control group, while the distribution of CK18, CK14, ATP1a1, and AQP5 protein in the cytoplasm of mesenchymal stem cells in printed blocks of TNF-α treatment group were significantly reduced in comparison. Conclusions: Exogenous TNF-α inhibits the directional differentiation of mesenchymal stem cells of mice into sweat gland cells in a three-dimensional environment, which may be related to the inhibition of the expression of Nanog mRNA and protein by TNF-α that subsequently results in the down-regulation of multi-directional differentiation potential of mesenchymal stem cells.

FGF-7 facilitates the process of psoriasis by inducing TNF-α expression in HaCaT cells.

The purpose of this study was to uncover the mechanism of tumor necrosis factor (TNF)-α induction by fibroblast growth factor-7 (FGF-7) in human HaCaT cells and the potential role of FGF-7-specific antibody F-9 in psoriatic therapy. TNF-α expression in HaCaT cells induced by FGF-7 was analyzed by quantitative polymerase chain reaction, western blot analysis, and enzyme-linked immunosorbent assays. In vivo, the BALB/c mouse psoriasis model established by topical application of imiquimod (IMQ) was used to determine the role of FGF-7-specific antibody (F-9) in skin inflammation. We found that induction of TNF-α expression by FGF-7 in HaCaT cells was suppressed by FGF-7-specific antibody F-9. Western blot analysis results showed that FGF-7 induced TNF-α expression in HaCaT cells via the FGF receptor 2 (FGFR2)/AKT/NF-κB signaling pathway. In vivo, F-9 could significantly ameliorate the inflammations in a mouse psoriatic model evaluated by Psoriasis Area and Severity Index scores and ear thickness, which was consistent with the results of hematoxylin-eosin staining, immunohistochemistry assay, and western blot analysis. These results indicate that FGF-7 induces TNF-α expression in HaCaT cells and FGF-7 antibody F-9 alleviates IMQ-induced psoriasiform in mice. Therefore, FGF-7/FGFR2 signaling pathway is a potential target for psoriasis treatment.

Acute stimulatory effect of tumor necrosis factor on the basolateral 50 pS K channels in the thick ascending limb of the rat kidney

The aim of the present study was to investigate the acute effect and mechanism of tumor necrosis factor (TNF) on basolateral 50 pS K channels in the thick ascending limb (TAL) of the rat kidney. The TAL tubules were isolated from the rat kidney, and the activity of the 50 pS K channels was recorded using the patch‑clamp technique. The results indicated that the application of TNF (10nM) significantly activated the 50 pS K channels and the TNF effect was concentration‑dependent. Inhibition of protein kinaseA, phospholipase A2 and protein tyrosine kinase using pathway inhibitors (H89, AACOCF3 and Herbimycin A, respectively) did not abolish the stimulatory effect of TNF, indicating that none of these pathways mediated the TNF effect. By contrast, the phenylarsine oxide inhibitor against protein tyrosine phosphatase (PTP) decreased the activity of the 50pS K channels and blocked the stimulatory effect of TNF on these channels. Furthermore, western blot analysis demonstrated that the application of TNF (10nM) in the TAL increased the phosphorylation of PTP, an indication of PTP activity stimulation. Thus, it was concluded that the acute application of TNF may stimulate the basolateral 50 pS K channel in the TAL and the stimulatory effect of TNF may be mediated by the PTP‑dependent pathway.

Pegylated liposomal‑paclitaxel induces ovarian cancer cell apoptosis via TNF‑induced ERK/AKT signaling pathway.

Ovarian cancer is one of the most common gynecological types of cancer and is characterized by a relatively high incidence and high mortality rate. Evidence has demonstrated that paclitaxel (PTX) is an effective therapeutic treatment for human ovarian cancer. In the present study, the inhibitory effects of pegylated liposomal (PL)-PTX on the growth of ovarian cancer cells were investigated in vitro; a CAOV-3-bearing mouse model was established to investigate the in vivo effects of PL-PTX on ovarian tumor growth. In the present study, the underlying mechanism of tumor necrosis factor (TNF)-induced inhibition of extracellular signal-regulated kinase (ERK)/protein kinase B (AKT) signaling pathway mediated by PL-PTX was analyzed within ovarian cancer cells. The results of the present study revealed that PL-PTX significantly inhibited the growth and aggressiveness of ovarian cancer cells in vitro and in vivo and apoptotic ability increased upon administration of PL-PTX. The expression levels of caspase-3/9 were significantly upregulated within PL-PTX-treated ovarian cancer cells. The expression and phosphorylation levels of ERK and AKT were markedly increased in response to PL-PTX treatment. In addition, the inhibitory effects of PL-PTX on ovarian cancer cells were eliminated by neutralizing antibodies against TNF. The observations of the present study revealed that PL-PTX induced ovarian cell apoptosis via the TNF-dependent pathway, which was significantly inhibited with the employment of antibodies against TNF. In vivo analysis demonstrated that PL-PTX treatment significantly inhibited ovarian tumor growth and prolonged the survival of tumor bearing mice. In conclusion, the findings of the present study have provided an insight into the potential mechanism of PL-PTX-induced apoptosis of ovarian cancer cells. As PL-PTX has been reported to induce ovarian tumor cell apoptosis via the TNF-induced ERK/AKT signaling pathway, PL-PTX may serve as an efficient anticancer drug for the treatment of ovarian cancer.

Lentivirus-mediated inhibition of tumour necrosis factor-α improves motor function associated with PRDX6 in spinal cord contusion rats.

The recovery of motor function in rats is inhibited following contusion spinal cord injury (cSCI). However, the mechanism of tumour necrosis factor α (TNF-α) in motor function after cSCI associated with peroxiredoxin 6 (PRDX6) remains unknown. We randomly divided rats into four groups: sham, cSCI, vector and lentivirus mediating TNF-α RNA interference (TNF-α-RNAi-LV) group. The Basso, Beattie, Bresnahan (BBB) scale was used to evaluate motor function. Real-time quantitative PCR (qRT-PCR) and western blotting were used to detect the expression of TNF-α and PRDX6, which were located in neurons using immunohistochemistry (IHC) and immunofluorescence. Subsequently, lentiviral-mediated TNF-α was used to determine the role of TNF-αand the relationship of PRDX6 and TNF-α in cSCI. After cSCI, the motor capability of hind limbs disappeared and was followed by recovery of function. IHC analysis indicated that TNF-α and PRDX6 were primarily located in spinal cord neurons. TNF-α interference significantly improved neural behaviour and increased expression of PRDX6. Our study suggests that inhibition of TNF-α can promote the recovery of motor function. The underlying mechanism of TNF-α-promoted motor function may be connected with the up-regulation of PRDX6. This provides a new strategy or target for the clinical treatment of SCI in future.

Mechanisms of tumor necrosis factor α antagonist-induced lupus in a murine model.

Tumor necrosis factor α (TNFα) antagonists are effective for treating rheumatoid arthritis and other inflammatory diseases, but their use can be complicated by the development of lupus-like phenomena. This study was undertaken to investigate the role of TNFα in a murine model of lupus. Toll-like receptor 7 (TLR-7) ligand-driven lupus was induced by injection of pristane into C57BL/6 (B6) mice deficient in TNFα (TNFα(-/-) ) or TNFα-intact B6 mice as wild-type controls. Autoantibody and type I interferon (IFN) production was measured in each group of mice, and the effects of the presence or absence of TNFα on type I IFN-producing plasmacytoid dendritic cells (PDCs), Ly-6C(high) monocytes, and TNFα-producing neutrophils were determined. TNFα(-/-) mice did not spontaneously develop autoantibodies or clinical manifestations of lupus, suggesting that TNFα deficiency alone is insufficient to cause lupus. Although the levels of type I IFN were comparable between untreated TNFα(-/-) and wild-type control mice, untreated TNFα(-/-) mice had increased circulating levels of PDCs and PDC-like cells, which enhanced the potential for production of type I IFN. When treated with pristane, TNFα(-/-) mice developed more severe lupus compared to pristane-treated controls, characterized by increased levels of anti-Sm/RNP autoantibodies, type I IFN, PDCs, and peritoneal inflammatory (Ly-6C(high) ) monocytes. In pristane-treated TNFα(-/-) mice, the numbers of neutrophils, a cell type that promotes resolution of inflammation, were decreased considerably, whereas the responses of inflammatory monocytes and PDCs and the production of type I IFN were increased and prolonged. Low levels of TNFα will increase the number of circulating PDCs in mice, thereby enhancing the potential to produce type I IFN. However, this does not necessarily lead to type I IFN production or autoimmunity unless there is concomitant exposure to endogenous TLR-7 ligands, which are released from dead cells following pristane treatment. In humans, the rate of clearance of dead cells, along with the levels of TNFα, may influence who will develop lupus following treatment with TNFα inhibitors.

Annexin A2 Regulates A Disintegrin and Metalloproteinase 17–mediated Ectodomain Shedding of Pro–Tumor Necrosis Factor-α in Monocytes and Colon Epithelial Cells

Understanding the mechanism of tumor necrosis factor (TNF)-α shedding is important because TNF-α triggers inflammatory bowel disease development. A disintegrin and metalloproteinase (ADAM) 17 is a key enzyme for the shedding of not only the type 1 membrane-anchored protein, amphiregulin, but also the type 2 protein, TNF-α. However, the detailed mechanism by which ADAM17 cleaves type 1 and 2 membrane-anchored proteins is unclear. Annexin (ANX) A2 is involved in ADAM17-mediated amphiregulin shedding. In this study, we examined whether ANX A2 is involved in TNF-α shedding. We prepared U937, HT29, and HCT116 cells overexpressing alkaline phosphatase (AP)-tagged proTNF-α and depleted ADAM17 and ANX A2. We assessed TNF-α release and shedding by measuring the TNF-α release concentration and AP activities in conditioned media after interleukin-1β or 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation by enzyme-linked immunosorbent assay and AP assay, respectively. A direct association of ANX A2 with ADAM17 was examined with immunoprecipitation and Western blotting. Enzyme-linked immunosorbent assay and AP assay showed interleukin-1β-induced TNF-α shedding in HCT116 and HT29 cells and TPA-induced TNF-α release in U937 cells. KB-R7785 and ADAM17 depletion significantly blocked TNF-α shedding by TPA. ANX A2 depletion significantly inhibited TNF-α shedding by interleukin-1β and TPA. In contrast, ANX A2 depletion did not abrogate ADAM17-mediated amphiregulin and heparin-binding epidermal growth factor-like growth factor shedding. ANX A2 was directly associated with ADAM17. ANX A2 was closely associated with ADAM17 and played an important role in TNF-α shedding by TPA. Inhibition of ANX A2 might be a new therapeutic strategy for prevention of TNF-α shedding during inflammatory bowel disease inflammation.

Acid-Induced Acute Lung Injury in Mice is Associated With p44/42 and c-Jun N-Terminal Kinase Activation and Requires the Function of Tumor Necrosis Factor α Receptor I

Aspiration of hydrochloric acid (HCl)-containing gastric juice leads to acute lung injury (ALI) and hypoxemic respiratory failure due to an exuberant inflammatory response associated with pulmonary edema from increased vascular and epithelial permeability. The aim of this study was to determine the role and signaling mechanisms of tumor necrosis factor α (TNF-α) in experimental ALI from HCl aspiration using a combination of genetic animal models and pharmacologic inhibition strategies. To this end, HCl was instilled intratracheally to mice, followed by respiratory system elastance measurement, bronchoalveolar lavage, and lung tissue harvesting 24 h after injection. Hydrochloric acid instillation induced an inflammatory response in the lungs of wild-type mice, evidenced as increased bronchoalveolar lavage total cells, neutrophils, and total protein; histologic lung injury score; and respiratory system elastance, whereas TNF-α receptor I mRNA levels were maintained. These alterations could be prevented by pretreatment with etanercept or genetic deletion of the 55-kd TNF-α receptor I, but not by deletion of the TNF-α gene. Hydrochloric acid induced a 6-fold increase in apoptotic, caspase 3-positive cells in lung sections from wild-type mice, which was abrogated in mice lacking TNF-α receptor I. In immunoblotting and immunohistochemistry studies, HCl stimulated signaling via p44/42 and c-Jun N-terminal kinase, which was blocked in TNF-α receptor I knockout mice. In conclusion, ALI induced by HCl requires TNF-α receptor I function and associates with activation of downstream proinflammatory signaling pathways p44/42 and c-Jun N-terminal kinase.

Epigallocatechin gallate–mediated protection against tumor necrosis factor- α–induced monocyte chemoattractant protein–1 expression is heme oxygenase–1 dependent

Flavonoids have been suggested to protect against atherosclerosis via their antioxidant and anti-inflammatory properties. Heme oxygenase–1 (HO-1) is an enzyme that plays an important role in the vascular system, and its induction may provide a protective role against atherosclerosis. We hypothesize that flavonoids can down-regulate endothelial inflammatory parameters by modulating HO-1–regulated cell signaling. We focused on the role of HO-1 and its major metabolic product, bilirubin, on mechanisms of tumor necrosis factor- α–induced endothelial cell activation and protection by the catechin epigallocatechin gallate (EGCG). Pretreatment with EGCG inhibited the secretion of monocyte chemoattractant protein–1 and the activation of activator protein–1 in porcine aortic endothelial cells stimulated with tumor necrosis factor– α. Moreover, EGCG up-regulated the expression of HO-1 and further induced the secretion of bilirubin. The observed anti-inflammatory effects of EGCG were mimicked by the HO-1 inducer cobalt protoporphyrin and abolished by HO-1 gene silencing. These data suggest that the protective properties of flavonoids, such as EGCG, against endothelial inflammation may be regulated in part though induction of HO-1 and subsequent activator protein–1 signaling.

Probiotic Lactobacillus reuteri suppress proinflammatory cytokines via c-Jun

Differential immunoregulatory capabilities of probiotic Lactobacillus were explored in the context of pediatric Crohn's disease. Experimental strategies addressed molecular mechanisms of tumor necrosis factor (TNF) suppression in activated macrophages by transcriptional regulation. Secreted factors produced by probiotic Lactobacillus reuteri strains were harvested and tested with human monocytes and macrophages. Quantitative immunoassays and real-time reverse-transcriptase polymerase chain reaction (RT-PCR) were used to examine relative quantities of human cytokines and TNF mRNA, respectively, and reporter assays assessed transcriptional regulation of TNF by probiotics. DNA-protein macroarrays interrogated probiotic-mediated effects on transcription factor activation. Finally, enzyme-linked immunosorbent assays (ELISAs) and immunoblots examined the involvement of the specific transcription factor AP-1 and its components. Probiotic L. reuteri strain ATCC PTA 6475 demonstrated the ability to potently suppress human TNF production by lipopolysaccharide-activated monocytes and primary monocyte-derived macrophages from children with Crohn's disease. Quantities of the chemokine MCP-1/CCL2 were also reduced by probiotic L. reuteri strain ATCC PTA 6475 in macrophages of children in remission. Quantitative real-time RT-PCR and luciferase reporter assays showed that transcriptional regulation of human TNF was a primary mechanism of probiotic-mediated immunomodulation. Probiotic L. reuteri suppressed TNF transcription by inhibiting activation of MAP kinase-regulated c-Jun and the transcription factor, AP-1. Human TNF and MCP-1 suppression by probiotic L. reuteri was strain-dependent, and the activation of c-Jun and AP-1 represent primary targets for probiotic-mediated suppression of TNF transcription. This report emphasizes the clonal nature of immunoprobiosis and delineation of a specific immunomodulatory mechanism for probiotic strain selection in future inflammatory bowel disease-oriented clinical trials.

Clinical studies with tumour necrosis factor.

The mechanism of tumour necrosis factor (TNF) cytotoxicity remains unknown. The in vivo antitumour effects of TNF may be related to direct cytotoxicity, immunomodulatory effects or endothelial effects on tumour vasculature. Phase I and early Phase II clinical trials of human recombinant TNF are under way in Japan, the USA, the UK and Germany. The maximum Phase II dose for TNF has not been established. The clinical toxicity of TNF is generally similar to that of other biological agents. Systemic toxicity, including fever, chills, anorexia and nausea, has been seen in most patients treated with TNF and has not been clearly related to dose. Other toxicities have included liver function abnormalities, hypotension, transient neurological changes and haematological abnormalities. Few clinical responses have been reported but organized Phase II testing remains to be completed. Combination trials with interferons have recently been initiated. Phase II efficacy studies of TNF as a single agent and in combination are needed for an assessment of the value of this agent in cancer therapy.

Tumor Necrosis Factor Blockade: Mechanism of Action

Modulation of the immune response with tumor necrosis factor (TNF) blockers is not a new treatment strategy for many inflammatory disorders; however, relatively little is known about their specific mechanism of action. Understanding the mode of action, pharmacology, and pharmacokinetics of the monoclonal TNF antibodies, infliximab and adalimumab, and the soluble TNF receptor, etanercept, may therefore enable us to account for their differing clinical profiles. The aim of this supplement is to explore the roles and mechanisms of TNF and TNF blockade using in vitro pharmacological and in vivo animal modeling experiments, and in vivo studies of the effects of etanercept on the inflammatory cascade in patients with psoriasis.

Hepatocyte-Specific IKKγ/NEMO Expression Determines the Degree of Liver Injury

NEMO is the regulatory subunit of the I kappa B kinase (IKK) complex and is involved in controlling nuclear factor kappaB (NF-kappaB) activation. NEMO knockout mice die during embryogenesis due to massive hepatocyte apoptosis. Here we investigated the role of NEMO-dependent signaling in hepatocytes during acute liver injury. We generated conditional hepatocyte-specific NEMO knockout mice using the loxP system with the Cre recombinase under the control of the albumin promoter (NEMODeltaLPC). In these mice, we studied mechanisms of tumor necrosis factor (TNF)- and ischemia/reperfusion-dependent liver cell damage. In adult NEMODeltaLPC animals, NEMO is specifically deleted in hepatocytes and no differences in survival, growth, and fertility were found when compared with wild-type (NEMO(f/f)) mice. TNF stimulation of NEMODeltaLPC mice resulted in high serum transaminase levels and massive hepatocyte apoptosis, which were associated with lack of I kappa B alpha degradation, inhibition of NF-kappaB activation, and target gene transcription. Additionally, ischemia/reperfusion resulted in higher nonparenchymal cell-dependent induction of oxidative stress and stronger inflammation in NEMODeltaLPC mice. This led to massive hepatocyte apoptosis and death of the animals, while NEMO(f/f) mice survived with significantly lesser liver damage, showing mainly necrotic cell death. Thus, complete inhibition of NF-kappaB activation in hepatocytes, in contrast to attenuation in hepatocyte-specific IKK2(-/-) mice, determines the type of liver cell damage during ischemia/reperfusion injury and is associated with a poor prognosis. Our results show that understanding of the fine tuning of NF-kappaB modulation during liver injury is essential to develop new therapeutic strategies.

LIGHT Signals Directly to Intestinal Epithelia to Cause Barrier Dysfunction via Cytoskeletal and Endocytic Mechanisms

LIGHT (lymphotoxin-like inducible protein that competes with glycoprotein D for herpes virus entry on T cells) is a tumor necrosis factor core family member that regulates T-cell activation and causes experimental inflammatory bowel disease. Additional data suggest that LIGHT may be involved in the pathogenesis of human inflammatory bowel disease. The aim of this study was to determine if LIGHT is capable of signaling directly to intestinal epithelia and to define the mechanisms and consequences of such signaling. The effects of LIGHT and interferon-gamma on barrier function, cytoskeletal regulation, and tight junction structure were assessed in mice and intestinal epithelial monolayers. LIGHT induced barrier loss in cultured epithelia via myosin II regulatory light chain (MLC) phosphorylation; both barrier loss and MLC phosphorylation were reversed by MLC kinase (MLCK) inhibition. Pretreatment with interferon-gamma, which induced lymphotoxin beta receptor (LT beta R) expression, was required for these effects, and neither barrier dysfunction nor intestinal epithelial MLC phosphorylation occurred in LT beta R knockout mice. In cultured monolayers, endocytosis of the tight junction protein occludin correlated with barrier loss. Internalized occludin colocalized with caveolin-1. LIGHT-induced occludin endocytosis and barrier loss were both prevented by inhibition of caveolar endocytosis. T cell-derived LIGHT activates intestinal epithelial LT beta R to disrupt barrier function. This requires MLCK activation and caveolar endocytosis. These data suggest a novel role for LIGHT in disease pathogenesis and suggest that inhibition of MLCK-dependent caveolar endocytosis may represent an approach to restoring barrier function in inflammatory bowel disease.

Tumor necrosis factor-induced nonapoptotic cell death requires receptor-interacting protein-mediated cellular reactive oxygen species accumulation.

The mechanism of tumor necrosis factor (TNF)-induced nonapoptotic cell death is largely unknown, although the mechanism of TNF-induced apoptosis has been studied extensively. In wild-type mouse embryonic fibroblast cells under a caspase-inhibited condition, TNF effectively induced cell death that morphologically resembled necrosis. In this study, we utilized gene knockout mouse embryonic fibroblasts cells and found that tumor necrosis factor receptor (TNFR) I mediates TNF-induced necrotic cell death, and that RIP, FADD, and TRAF2 are critical components of the signaling cascade of this TNF-induced necrotic cell death. Inhibitors of NF-kappaB facilitated TNF-induced necrotic cell death, suggesting that NF-kappaB suppresses the necrotic cell death pathway. JNK, p38, and ERK activation seem not to be required for this type of cell death because mitogen-activated protein kinase inhibitors did not significantly affect TNF-induced necrotic cell death. In agreement with the previous reports that the reactive oxygen species (ROS) may play an important role in this type of cell death, the ROS scavenger butylated hydroxyanisole efficiently blocked TNF-induced necrotic cell death. Interestingly, during TNF-induced necrotic cell death, the cellular ROS level was significantly elevated in wild type, but not in RIP(-/-), TRAF2(-/-), and FADD(-/-) cells. These results suggest that RIP, TRAF2, and FADD are crucial in mediating ROS accumulation in TNF-induced necrotic cell death.