Wild-type TNF-α Research Articles

The Association of HHV-6 and the TNF-α (-308G/A) Promotor with Major Depressive Disorder Patients and Healthy Controls in Thailand.

Major depressive disorder (MDD) is a silent global health problem that can lead to suicide. MDD development is suggested to result from numerous risk factors, including genetic factors. A precise tool for MDD diagnosis is currently not available. Recently, inflammatory processes have been identified as being strongly involved in MDD development and the reactivation of human herpesvirus type 6 (HHV-6), upregulating cytokines such as TNF-α, which are associated with MDD. Therefore, this study aimed to determine the association of HHV-6 with genetic factors, especially TNF-α mutation, in MDD patients and their relatives compared to healthy controls. The Patient Health Questionnaire (PHQ-9) was used to evaluate MDD status, and 471 oral buccal samples were investigated for HHV-6 infection and viral copy number by qPCR. TNF-α (-308G/A) gene mutation and the cytokines TNF-α, IL-6, and IL-10 were analyzed by high-resolution melting (HRM) analysis and enzyme-linked immunosorbent assay (ELISA). Whole-exome sequencing of buccal samples was performed to analyze for genetic factors. The results showed significantly higher HHV-6 positivities and viral loads in MDD patients (15/59 (25.67%) and 14,473 ± 16,948 copies/µL DNA) and their relatives (blood relatives 17/36 (47.22%) and 8146 ± 5656 copies/µL DNA); non-blood relatives 7/16 (43.75%) and 20,721 ± 12,458 copies/µL DNA) compared to the healthy population (51/360 (14.17%) and 6303 ± 5791 copies/µL DNA). The TNF-α (-308G/A) mutation showed no significant difference. Surprisingly, 12/26 (46.15%) participants with the TNF-α (-308G/A) mutation showed HHV-6 positivities at higher rates than those with wild-type TNF-α (-308G) (70/267 (26.22%)). HHV-6-positive participants with TNF-α (-308G/A) showed higher levels of TNF-α, IL-6, and IL-10 than those of negative control. Exome analysis revealed that common mutations in immune genes were associated with depression. Therefore, this study unveiled the novel association of inflammatory gene TNF-α (-308G/A) mutations with HHV-6 reactivation, which could represent a combined risk factor for MDD. This result could induce further research on MDD development and clinical applications.

Smooth Muscle LRRC8A Anion Channel Knockout Promotes Vasodilation and Protects Against TNFα‐induced Vascular Dysfunction

Tumor necrosis factor‐α (TNFα), a pro‐inflammatory cytokine, is associated with cardiovascular risk. Volume‐regulated anion channels (VRACs) are activated by TNFα and are composed of Leucine Rich Repeat Containing 8 (LRRC8) family proteins (LRRC8A, B, C, D and E). LRRC8A is required for all VRACs. We previously determined that VRAC inhibitors or LRRC8A knockdown suppress the inflammatory response to TNFα in cultured vascular smooth muscle cells, however, the role of these channels in vasomotor function has not been studied. We hypothesized that loss of LRRC8A would prevent the impairment of vascular function by TNFα. We created vascular smooth muscle‐specific (Sm22alpha‐Cre) LRRC8A knockout (KO) mice and isolated mesenteric arteries from male mice for wire myograph recording. Vessel segments were incubated with TNFα (10 ng/mL) or vehicle for 2 days in tissue culture. Contractile responses to norepinephrine (NE) were unaltered in LRRC8A KO mice but enhanced by TNFα in wild type (WT) than in KO rings (WT+TNFα 284 ± 18.9 %, KO+TNFα 222 ± 7.1 % of KCl contraction, *p<0.05, n = 5‐6). In the vehicle‐treated group both endothelium‐dependent (acetylcholine, ACh) and ‐independent (sodium nitroprusside, SNP) relaxation were increased in LRRC8A KO compared to WT rings contracted with phenylephrine (PE). Exposure to TNFα impaired relaxation to both ACh and SNP in WT vessels but had no effect on relaxation of LRRC8A KO vessels (black *p<0.05 compared to WT control, red *p<0.05 compared to TNFα‐treated, n = 6‐8). The enhanced relaxation responses of KO vessels were recapitulated in WT rings by 20 min of exposure to a VRAC inhibitor, carbenoxolone (CBX; 100 μM). Thus, in addition to its established role in TNFα signaling, LRRC8A anion channel activity modulates vascular reactivity with loss of function enhancing vasodilator function. VRAC inhibition may provide a novel therapeutic approach to the prevention and treatment of inflammation‐induced vascular dysfunction.

Delicate regulation of IL-1β-mediated inflammation by cyclophilin A.

The inflammatory response is tightly regulated, but its regulatory principles are still incompletely understood. Cyclophilin A (CypA) has long been considered as a pro-inflammatory factor. Here, we discover how CypA precisely regulates interleukin-1β (IL-1β)-mediated inflammatory responses. In lipopolysaccharide-treated mice, CypA deficiency initially inhibits and then promotes lung inflammation, which is closely related to IL-1β production. Mechanistically, CypA not only facilitates pro-IL-1β processing by increasing Smurf1-mediated K63-linked ubiquitination in an ATP-dependent manner but also accelerates pro-IL-1β degradation, depending on Smurf1-mediated K48-linked ubiquitination. Moreover, in IL-1β-treated mice, CypA exacerbates lung injury by enhancing cytokine production. It also upregulates the ILK/AKT pathway by inhibiting Cyld-mediated K63-linked ILK deubiquitination, which promotes the epithelial-mesenchymal transition (EMT) to facilitate lung repair. Collectively, CypA promotes inflammation activation by increasing IL-1β production and then promotes inflammation resolution by enhancing redundant pro-IL-1β degradation and IL-1β-induced EMT, indicating the complex and delicate regulation of inflammatory response.

Gas6 Inhibits Toll-Like Receptor-Mediated Inflammatory Pathways in Mouse Microglia via Axl and Mer.

Background: Microglia are well known key regulators of neuroinflammation which feature in multiple neurodegenerative disorders. These cells survey the CNS and, under inflammatory conditions, become “activated” through stimulation of toll-like receptors (TLRs), resulting in changes in morphology and production and release of cytokines. In the present study, we examined the roles of the related TAM receptors, Mer and Axl, and of their ligand, Gas6, in the regulation of microglial pro-inflammatory TNF-α production and microglial morphology.Methods: Primary cultures of murine microglia of wild-type (WT), Mer−/− and Axl−/− backgrounds were stimulated by the TLR4 agonist, lipopolysaccharide (LPS) with or without pre-treatment with Gas6. Gene expression of TNF-α, Mer, and Axl was examined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA) was used to measure TNF-α release from microglia. Immunofluorescence staining of β-actin and the microglial marker Iba1 was performed to reveal microglial morphological changes, with cellular characteristics (area, perimeter, Feret’s diameter, minimum Feret, roundness, and aspect ratio) being quantified using ImageJ software.Results: Under basal conditions, TNF-α gene expression was significantly lower in Axl−/− microglia compared to WT cells. However, all microglial cultures robustly responded to LPS stimulation with the upregulation of TNF-α expression to similar degrees. Furthermore, Mer receptor expression was less responsive to LPS stimulation when in Axl knockout cells. The presence of Gas6 consistently inhibited the LPS-induced upregulation of TNF-α in WT, Mer−/− and Axl−/− microglia. Moreover, Gas6 also inhibited LPS-induced changes in the microglial area, perimeter length, and cell roundness in wild-type cells.Conclusion: Gas6 can negatively regulate the microglial pro-inflammatory response to LPS as well as via stimulation of other TLRs, acting through either of the TAM receptors, Axl and Mer. This finding indicates an interaction between TLR and TAM receptor signaling pathways and reveals an anti-inflammatory role for the TAM ligand, Gas6, which could have therapeutic potential.

Effects of A94T and P84L Polymorphisms Within the TNF-α Gene on Proliferation and Activation of Hepatic Stellate Cells.

Tumor necrosis factor alpha (TNF-α) appears to play an important role in proliferation and activation of hepatic stellate cells (HSCs), but it is unclear whether single nucleotide polymorphisms (SNPs) in the TNF-α gene influence HSCs function. In this study, we explored the effects of TNF-α A94T and P84L polymorphisms on the level of TNF-α, proliferation and activation of HSCs. It was found that A94T and P84L SNPs of TNF-α downregulated the mRNA and protein level of TNF-α in recombinant cells. Compared with wild-type TNF-α, A94T and P84L SNPs could decrease the growth or activation inhibitory effects of TNF-α on LX-2 cells, the human HSC line. In addition, A94T SNPs were associated with significantly lower expression of matrix-metalloproteinase 2 (MMP 2) or 9, but P84L SNP only decreased the mRNA level of MMP 9. A94T and P84L SNPs of TNF-α downregulated the level of IL-6. Furthermore, A94T and P84L SNPs decrease the activation inhibitory effects of TNF-α on LX-2 cells through inhibiting the phosphorylation levels of inhibitory kappa B-alpha (IκB-α) and P65. This study provides two vital SNPs for further functional or case-control studies of TNF-α SNPs.

Single nucleotide polymorphisms in ATM, TNF-α and IL6 genes and risk of radiotoxicity in breast cancer patients

Although oncological therapies have improved in the last decades, breast cancer (BC) remains a serious health problem worldwide. Radiotherapy (RT) is one of the most frequently used treatments for cancer aimed at eliminating tumor cells. However, it can also alter the surrounding normal tissue, especially the skin, and patient reactions may vary as a result of extrinsic and intrinsic factors. We evaluated the association of gene polymorphisms ATM Asp1853Asn, IL-6 G-174C and TNF-α G-308A involved in central phenotype pathways and development of individual radiosensitivity in BC patients with an exacerbated response to RT. Although univariate analysis results did not show a significant association with this trait, the interaction analysis between polymorphisms showed an increased risk of patients presenting wild-type TNF-α G-308A genotype and mutant IL-6 G-174C genotype, and heterozygous TNF-α G-308A genotype and heterozygous IL-6G-174C genotype. On the other hand, our results showed that breast size and patient age influenced the determination of RT-associated effects. Considering that the trait is multifactorial, other significant elements for the determination of individual radiosensitivity should be considered, together with the establishment of specific polymorphic variants.

RIPK3/MLKL-Mediated Neuronal Necroptosis Modulates the M1/M2 Polarization of Microglia/Macrophages in the Ischemic Cortex.

Cell death and subsequent inflammation are 2 key pathological changes occurring in cerebral ischemia. Active microglia/macrophages play a double-edged role depending on the balance of their M1/M2 phenotypes. Necrosis is the predominant type of cell death following ischemia. However, how necrotic cells modulate the M1/M2 polarization of microglia/macrophages remains poorly investigated. Here, we reported that ischemia induces a rapid RIPK3/MLKL-mediated neuron-dominated necroptosis, a type of programmed necrosis. Ablating RIPK3 or MLKL could switch the activation of microglia/macrophages from M1 to the M2 type in the ischemic cortex. Conditioned medium of oxygen-glucose deprivation (OGD)-treated wild-type (WT) neurons induced M1 polarization, while that of RIPK3−/− neurons favored M2 polarization. OGD treatment induces proinflammatory IL-18 and TNFα in WT but not in RIPK3−/− neurons, which in turn upregulate anti-inflammatory IL-4 and IL-10. Furthermore, the expression of Myd88—a common downstream adaptor of toll-like receptors—is significantly upregulated in the microglia/macrophages of ischemic WT but not of RIPK3−/− or MLKL−/− cortices. Antagonizing the function of Myd88 could phenocopy the effects of RIPK3/MLKL-knockout on the polarization of microglia/macrophages and was neuroprotective. Our data revealed a novel role of necroptotic neurons in modulating the M1/M2 balance of microglia/macrophages in the ischemic cortex, possibly through Myd88 signaling.

Polymorphisms of TLR4 Asp299Gly and TNF-α -308G/A in Leptospirosis

AbstractBackground : TLR4 Asp299Gly and TNF-α -308G/A polymorphisms have been shown to be associated with increased susceptibility and severity of infection. TLR4 Asp299Gly polymorphism could affect the host’s ability to respond to leptospira sp. TNF-α -308G/A polymorphism, is associated with the high producer of TNF-α.Methods : Total of 36 leptospirosis patients (IgM anti leptospira and MAT positive) and healthy individual with the equal number were included. The polymorphisms were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using site spesific restriction enzyme.Results : Distribution of homozygous wild-type TLR4 Asp299Gly polymorphism was higher in both of groups ( 94.5:97.2%.) and homozygous mutant allele was absent. There was not significantly difference of TLR4 Asp299Gly in leptospirosis patients and healthy group ( ρ=1.00; OR 0.5; 95%CI, 0.04-5.6) and between mild and severe leptospirosis (ρ=0.54; OR 1.54 ; 95% CI, 1.20-1.98). The presence of homozygous wild-type TNF-α -308G/A polymorphism was higher between leptospirosis patients and healthy group (100:94.5%) andhomozygous mutant allele was not found in both of the groups. No significantly different of TNF-α -308G>A polymorphism between leptospirosis patient and healthy group (ρ=0.49).Conclusions : In this study, the polymorphisms of TLR4 Asp299Gly and TNF-α -308G/A were not associated with the susceptibility and severity of leptospirosis. Keywords : Leptospirosis, TLR4 Asp299Gly polymorphism, TNF-α -308G/A polymorphism

Estrogen receptor α (ESR1) over-expression mediated apoptosis in Hep3B cells by binding with SP1 proteins

Previous studies have reported that estrogen receptors (ERs) are expressed in normal human liver, chronic hepatitis, and benign hepatic tumor tissues. However, decreased expression of ERs can be observed in hepatocellular carcinoma (HCC) and the role of ERs in HCC is not fully understood. Thus, the present study aimed to investigate the molecular mechanism induced by the overexpression of ERα (ERα (ESR1)) in Hep3B cells. We first detected the induction of apoptosis in ER-negative Hep3B cells using DNA fragmentation assay and flow cytometry. We found that ERα and ERα plus 17β-estradiol treatment increased apoptosis in Hep3B cells. Additionally, western blotting showed increased expression of active caspase 3 and tumor necrosis factor α (TNFα (TNF)) in ERα-transfected cells. To further understand the importance of SP1-binding sites in the TNFα promoter, ERα-negative Hep3B cells were co-transfected with ERα and a wild-type TNFα plasmid or TNFα with deleted SP1 regions. Deletion of both distant and primal SP1 sites abolished the activity of ERα, and similar results were observed by blocking the expression of SP1 protein using mithramycin (MA). This result indicates that SP1 protein is essential for ERα-activated TNFα promoter activity. Co-immunoprecipitation assay further confirmed the binding interaction between ERα and SP1 in a ligand-dependent manner. In general, we demonstrate that the overexpression of ERα mediates apoptosis in ERα-negative Hep3B cells by the binding of ERα to SP1 protein. Additionally, this ERα-SP1 complex binds to the proximal and distal sites of the TNFα gene promoter and further induces the expression of active caspase 3 in a ligand-dependent manner.

Role of miR-19a targeting TNF-α in mediating ulcerative colitis

Objective. Ulcerative colitis (UC) is a widely studied inflammatory disease associated with differential expression of genes involved in immune function, wound healing, and tissue remodeling. MicroRNAs have been reported to play a role in various cancer types. However, the mechanism of how microRNAs regulate UC remains unclear. Methods.In the present study, we investigated the role of miR-19a and tumor necrosis factor (TNF)-α in human colon tissues with UC and dextran sodium sulfate (DSS)-induced experimental colitis. Results. We identified that the expression of miR-19a was significantly reduced and TNF-α was remarkably increased in human colon tissue with UC. Moreover, this observation of miR-19a and TNF-α was also occurred in DSS-treated mice colitis. Further, we observed that miR-19a directly regulated TNF-α expression because miR-19a can suppress the expression of wild-type TNF-α reporter, but not the mutant form. The expression of inflammatory factors TNF-α, IL-8, and GM-GSF were significantly elevated upon application of miR-19a inhibitor. Conclusion. Taken together, this study determines the levels of miR-19a and TNF-α in both DSS-induced experimental murine colitis and human UC and further demonstrates that miR-19a might directly regulate TNF-α. The findings may provide a new insight in the clinical treatment of UC.

Protein Phosphatase 2A Catalytic Subunit α Plays a MyD88-Dependent, Central Role in the Gene-Specific Regulation of Endotoxin Tolerance

MyD88, the intracellular adaptor of most TLRs, mediates either proinflammatory or immunosuppressive signaling that contributes to chronic inflammation-associated diseases. Although gene-specific chromatin modifications regulate inflammation, the role of MyD88 signaling in establishing such epigenetic landscapes under different inflammatory states remains elusive. Using quantitative proteomics to enumerate the inflammation-phenotypic constituents of the MyD88 interactome, we found that in endotoxin-tolerant macrophages, protein phosphatase 2A catalytic subunit α (PP2Ac) enhances its association with MyD88 and is constitutively activated. Knockdown of PP2Ac prevents suppression of proinflammatory genes and resistance to apoptosis. Through site-specific dephosphorylation, constitutively active PP2Ac disrupts the signal-promoting TLR4-MyD88 complex and broadly suppresses the activities of multiple proinflammatory/proapoptotic pathways as well, shifting proinflammatory MyD88 signaling to a prosurvival mode. Constitutively active PP2Ac translocated with MyD88 into the nuclei of tolerant macrophages establishes the immunosuppressive pattern of chromatin modifications and represses chromatin remodeling to selectively silence proinflammatory genes, coordinating the MyD88-dependent inflammation control at both signaling and epigenetic levels under endotoxin-tolerant conditions.

Microglial p38α MAPK is critical for LPS-induced neuron degeneration, through a mechanism involving TNFα

BackgroundThe p38α MAPK isoform is a well-established therapeutic target in peripheral inflammatory diseases, but the importance of this kinase in pathological microglial activation and detrimental inflammation in CNS disorders is less well understood. To test the role of the p38α MAPK isoform in microglia-dependent neuron damage, we used primary microglia from wild-type (WT) or p38α MAPK conditional knockout (KO) mice in co-culture with WT cortical neurons, and measured neuron damage after LPS insult.ResultsWe found that neurons in co-culture with p38α-deficient microglia were protected against LPS-induced synaptic loss, neurite degeneration, and neuronal death. The involvement of the proinflammatory cytokine TNFα was demonstrated by the findings that p38α KO microglia produced much less TNFα in response to LPS compared to WT microglia, that adding back TNFα to KO microglia/neuron co-cultures increased the LPS-induced neuron damage, and that neutralization of TNFα in WT microglia/neuron co-cultures prevented the neuron damage. These results using cell-selective, isoform-specific KO mice demonstrate that the p38α MAPK isoform in microglia is a key mediator of LPS-induced neuronal and synaptic dysfunction. The findings also provide evidence that a major mechanism by which LPS activation of microglia p38α MAPK signaling leads to neuron damage is through up-regulation of the proinflammatory cytokine TNFα.ConclusionsThe data suggest that selective targeting of p38α MAPK signaling should be explored as a potential therapeutic strategy for CNS disorders where overproduction of proinflammatory cytokines is implicated in disease progression.

Relation of Serum TNF-α and TNF-α Genotype with Delayed Cerebral Ischemia and Outcome in Subarachnoid Hemorrhage

The pathogenesis of delayed cerebral ischemia (DCI) after subarachnoid hemorrhage (SAH) remains obscure. The authors assessed the relationship of tumor necrosis factor alpha (TNF-α) and TNF-α gene polymorphisms with occurrence of DCI and poor outcome at 3months. Serum levels of TNF-α were measured every other day until discharge in 67 patients and the mean serum levels per patient during days 0-12 were dichotomized at the median value of the whole group. TNF-α genotyping was available in 31 patients and related to serum TNF-α by means of one-way ANOVA analysis. The authors calculated hazard ratio's (HR) with corresponding 95% confidence intervals (CI) for the association with DCI by means of Cox proportional hazard analysis and odds ratio's (OR) for the association with poor clinical outcome by means of logistic regression analysis. In both analyses the authors adjusted for sex, age, amount of blood, and clinical condition at admission. Leukocytes and CRP were investigated similarly for comparison. For high-serum TNF-α levels during days 0-12 adjusted HR was 0.6 (95%CI: 0.1-2.4) for DCI and adjusted OR 2.0 (95%CI: 0.4-9.0) for clinical outcome. Serum TNF-α levels were 11.4pg/ml for wildtype TNF-α genotype and 9.7pg/ml for the non-wildtype TNF-α genotype (P=0.15). For the non-wildtype TNF-α genotype the HR for DCI was 0.4 (95%CI: 0.1-2.6) and the OR for clinical outcome was 0.8 (95%CI: 0.1-4.0). It is unlikely that serum TNF-α or TNF-α genotype play an important role in the occurrence of DCI after SAH.

Leader sequence is required for activity of transmembrane tumor necrosis factor-α

Transmembrane tumor necrosis factor α (tmTNF-α) has a variety of biological activities different from soluble TNF-α (sTNF-α), but the only difference in sequence is its leader sequence (LS). To investigate the effect of the LS on tmTNF-α activity, single amino acid substitutions in the LS and its linked extracellular mature domain were made in an in vitro translation system and in an intact cell system. Mutations at Met −71 and Cys −28 in the LS obliterated cytotoxicity of tmTNF-α, whilst their secretory form retained full activity compared to parental sTNF-α. The lost cytotoxicity of Met −71 mutant tmTNF-α was partly due to a reduced receptor binding activity. In spite of full receptor binding activity, Cys −28 mutant tmTNF-α failed to induce NO production and iNOS mRNA transcription via forward signaling, but synergized with sTNF-α in IL-8 mRNA transcription via reverse signaling. The Asp 143 mutant tmTNF-α lost the ability to bind TNFR and to kill MCF-7 cells, whilst its secretory form retained about 60% cytotoxicity of parental sTNF-α. Although the mutation at Phe 87 had full activity in both forms, its membrane form induced a change in cell death mode from apoptosis to necrosis, in contrast to wild-type TNF-α whose membrane molecule chiefly induced apoptosis and secretory molecule mainly caused necrosis in MCF-7, respectively. The data suggest that the LS may be required for maintaining the correct structure and the bioactivity of tmTNF-α.

The use of a mutant TNF-α as a vaccine adjuvant for the induction of mucosal immune responses

Safe and potent adjuvants are required in order to establish effective mucosal vaccines. Cytokines are promising adjuvants because they are human-derived safe biomaterial and display immune-modulating functions. We have created a mutant tumor necrosis factor-α (TNF-α), mTNF-K90R, that exhibits high bioactivity and resistance to proteases. Here, we examined the potential of mTNF-K90R as a mucosal adjuvant. Initially, we showed that intranasal co-administration of mTNF-K90R with ovalbumin (OVA) potently produced OVA-specific Immunoglobulin (Ig) G antibodies (Abs) in serum and IgA Abs both at local and distal mucosal sites compared to co-administration with wild-type TNF-α. The OVA-specific immune response was characterized by high levels of serum IgG1 and increased production of interleukin-4 (IL-4), IL-5 and IL-10 from splenocytes of immunized mice, suggesting a Th2 response. Furthermore, intranasal immunization with an antigen from influenza virus plus mTNF-K90R exhibited mucosal adjuvant activity for induction of both systemic and mucosal immune responses. Importantly, histopathological examination of the nasal tissue of mTNF-K90R treated mice detected no signs of toxicity. These findings suggest that mTNF-K90R is safe and effective mucosal adjuvant and this system may have potential application as a universal mucosal adjuvant system for mucosal vaccines improving the immune response to a variety of viral antigens.

MTOR‐dependent IL‐10 expression inhibits LPS induction of tissue factor and cytokines in macrophages.

Bacterial lipopolysaccharide (LPS) induces monocytes/macrophages to express proinflammatory cytokines and tissue factor (TF), the primary activator of the coagulation cascade. Anti‐inflammatory signaling pathways including the phosphatidylinositol‐3‐kinase (PI3K)‐Akt pathway inhibit proinflammatory gene expression in macrophages. We determined whether the mammalian target of rapamycin (mTOR) and induction of the anti‐inflammatory cytokine interleukin‐10 (IL‐10) inhibited LPS‐induced proinflammatory and TF gene expression in peritoneal macrophages (PMs) in a PI3K‐Akt‐dependent manner. We used wild type (WT) peritoneal macrophages (PMs), and PMs from PTENflox/flox/LysMCre mice (PTEN−/− PMs), which have increased Akt activity. LPS induction of IL‐10 mRNA and protein was increased in PTEN−/− PMs compared to WT PMs. Pharmacologic inhibition of mTOR with rapamycin inhibited LPS induction of IL‐10 mRNA and protein, and enhanced the expression of TF and the proinflammatory cytokines interleukin‐6 (IL‐6) and tumor necrosis factor‐α (TNFα). Neutralization of endogenous IL‐10 also enhanced LPS induction of TNFα, IL‐6 and TF expression in WT and PTEN−/− PMs. The results indicate that mTOR‐dependent IL‐10 expression leads to inhibition of LPS induction of TF and the proinflammatory cytokines IL‐6 and TNFα in WT macrophages

N-cadherin as a predictor of brain metastases in NSCLC

Tumor necrosis factor α (TNFα) enhances proliferation of chemically-induced mammary tumors and of T47D human cell line through not fully understood pathways. Here, we explored the intracellular signaling pathways triggered by TNFα, the participation of TNFα receptor (TNFR) 1 and TNFR2 and the molecular mechanism leading to breast cancer growth. We demonstrate that TNFα induced proliferation of C4HD murine mammary tumor cells and of T47D cells through the activation of p42/p44 MAPK, JNK, PI3-K/Akt pathways and nuclear factor-kappaB (NF-κB) transcriptional activation. A TNFα-specific mutein selectively binding to TNFR1 induced p42/p44 MAPK, JNK, Akt activation, NF-κB transcriptional activation and cell proliferation, just like wild-type TNFα, while a mutein selective for TNFR2 induced only p42/p44 MAPK activation. Interestingly, blockage of TNFR1 or TNFR2 with specific antibodies was enough to impair TNFα signaling and biological effect. Moreover, in vivo TNFα administration supported C4HD tumor growth. We also demonstrated, for the first time, that injection of a selective inhibitor of NF-κB activity, Bay 11-7082, resulted in regression of TNFα-promoted tumor. Bay 11-7082 blocked TNFα capacity to induce cell proliferation and up-regulation of cyclin D1 and of Bcl-xL in vivo and in vitro. Our results reveal evidence for TNFα as a breast tumor promoter, and provide novel data for a future therapeutic approach using TNFα antagonists and NF-κB pharmacological inhibitors in established breast cancer treatment.

TNFα acting on TNFR1 promotes breast cancer growth via p42/P44 MAPK, JNK, Akt and NF-κB-dependent pathways

Tumor necrosis factor α (TNFα) enhances proliferation of chemically-induced mammary tumors and of T47D human cell line through not fully understood pathways. Here, we explored the intracellular signaling pathways triggered by TNFα, the participation of TNFα receptor (TNFR) 1 and TNFR2 and the molecular mechanism leading to breast cancer growth. We demonstrate that TNFα induced proliferation of C4HD murine mammary tumor cells and of T47D cells through the activation of p42/p44 MAPK, JNK, PI3-K/Akt pathways and nuclear factor-kappaB (NF-κB) transcriptional activation. A TNFα-specific mutein selectively binding to TNFR1 induced p42/p44 MAPK, JNK, Akt activation, NF-κB transcriptional activation and cell proliferation, just like wild-type TNFα, while a mutein selective for TNFR2 induced only p42/p44 MAPK activation. Interestingly, blockage of TNFR1 or TNFR2 with specific antibodies was enough to impair TNFα signaling and biological effect. Moreover, in vivo TNFα administration supported C4HD tumor growth. We also demonstrated, for the first time, that injection of a selective inhibitor of NF-κB activity, Bay 11-7082, resulted in regression of TNFα-promoted tumor. Bay 11-7082 blocked TNFα capacity to induce cell proliferation and up-regulation of cyclin D1 and of Bcl-x L in vivo and in vitro. Our results reveal evidence for TNFα as a breast tumor promoter, and provide novel data for a future therapeutic approach using TNFα antagonists and NF-κB pharmacological inhibitors in established breast cancer treatment.

THE TUMOR NECROSIS FACTOR α −308 G/A POLYMORPHISM DOES NOT INFLUENCE INFLAMMATION AND COAGULATION RESPONSE IN HUMAN ENDOTOXEMIA

Tumor necrosis factor (TNF)-alpha plays a major role in the immune system. Release of proinflammatory cytokines, such as TNF-alpha and interleukin 6, by macrophages and other cells occurs in response to bacterial products. It has been reported that the TNF-alpha -308 G/A polymorphism in the TNF-alpha gene determines basal TNF-alpha levels. We hypothesized that it may also be associated with the degree of inflammatory response in a well-standardized model of systemic inflammation. Eighty-seven young men (age range, 19-35 years) received 2 ng/kg i.v. endotoxin (lipopolysaccharide [LPS]). The TNF-alpha promoter genotype was analyzed on a TaqMan genomic analyzer. Inflammation markers (interleukin 6, TNF-alpha), temperature, and coagulation markers (prothrombin fragment F1+2, D-dimer) were measured at 0, 2, 6, and 24 h after LPS infusion. Tumor necrosis factor-alpha plasma concentrations increased from a baseline 1.3 ng/L (range, 0.8-3.1 ng/L) before LPS infusion to a peak of 57.5 ng/L (range, 10.8-131.4 ng/L) at 2 h after LPS and then decreased continually to 10.8 ng/L (range, 4.7-16.5 ng/L) after 6 h and returned to baseline values after 24 h (1.9 ng/L [range, 1.1-3.9 ng/L]). We observed no significant differences in TNF-alpha baseline levels or in response to LPS after stratification of the data according to TNF-alpha genotype. Basal and peak values of selected inflammatory and coagulation markers were not different between wild-type TNF-alpha -308 individuals (GG) and carriers of the TNF-alpha -308 mutant allele (GA and AA). The TNF-alpha -308 G/A polymorphism does not contribute significantly to the individual variability of systemic TNF-alpha plasma concentrations after endotoxin challenge. Thus, if any, the impact of the TNF-alpha -308 G/A polymorphism on systemic endotoxin-triggered inflammation seems to be limited.

Astrocyte reactivity to Fas activation is attenuated in TIMP-1 deficient mice, an in vitrostudy

BackgroundTissue inhibitor of metalloproteinases-1 (TIMP-1) is a multifunctional secreted protein with pleiotropic actions, including the inhibition of matrix metalloproteinases (MMPs), cell death/survival and growth promoting activities. After inflammatory challenge, the levels of TIMP-1 are highly and selectively upregulated in astrocytes among glial cells, but little is know about its role in these neural cells. We investigated the influence of TIMP-1 null mutation in the reactivity of cultured astrocytes to pro-inflammatory stimuli with TNF-α and anti-Fas antibody.ResultsWhen compared to WT, mutant astrocytes displayed an overall increased constitutive gelatinase expression and were less responsive to Fas-mediated upregulation of MMP-9, of monocyte chemoattractant protein-1 (MCP-1) and of intercellular cell adhesion molecule-1 (ICAM-1), all markers of astrocyte inflammatory response. In contrast, TNF-α treatment induced all these factors similarly regardless of the astrocyte genotype. The incorporation of 3H-thymidin, a marker of cell proliferation, increased in wild-type (WT) astrocytes after treatment with anti-Fas antibody or recombinant TIMP-1 but not in mutant astrocytes. Finally, lymphocyte chemotaxis was differentially regulated by TNF-α in WT and TIMP-1 deficient astrocytes.ConclusionWe provide evidence that the alteration of the MMP/TIMP balance in astrocytes influences their reactivity to pro-inflammatory stimuli and that Fas activation modulates the expression of members of the MMP/TIMP axis. We hypothesise that the Fas/FasL transduction pathway and the MMP/TIMP system interact in astrocytes to modulate their inflammatory response to environmental stimuli.