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Mesenchymal stem cells (MSCs) are used in cell therapy; nonetheless, their application is limited by their poor survival after transplantation in a proinflammatory microenvironment. Macroautophagy/autophagy activation in MSCs constitutes a stress adaptation pathway, promoting cellular homeostasis. Our proteomics data indicate that RUBCNL/PACER (RUN and cysteine rich domain containing beclin 1 interacting protein like), a positive regulator of autophagy, is also involved in cell death. Hence, we screened MSC survival upon various cell death stimuli under loss or gain of function of RUBCNL. MSCs were protected from TNF (tumor necrosis factor)-induced regulated cell death when RUBCNL was expressed. TNF promotes inflammation by inducing RIPK1 kinase-dependent apoptosis or necroptosis. We determine that MSCs succumb to RIPK1 kinase-dependent apoptosis upon TNF sensing and necroptosis when caspases are inactivated. We show that RUBCNL is a negative regulator of both RIPK1-dependent apoptosis and necroptosis. Furthermore, RUBCNL mutants that lose the ability to regulate autophagy, retain their function in negatively regulating cell death. We also found that RUBCNL forms a complex with RIPK1, which disassembles in response to TNF. In line with this finding, RUBCNL expression limits assembly of RIPK1-TNFRSF1A/TNFR1 complex I, suggesting that complex formation between RUBCNL and RIPK1 represses TNF signaling. These results provide new insights into the crosstalk between the RIPK1-mediated cell death and autophagy machineries and suggest that RUBCNL, due to its functional duality in autophagy and apoptosis/necroptosis, could be targeted to improve the therapeutic efficacy of MSCs. Abbreviations: BAF: bafilomycin A1; CASP3: caspase 3; Caspases: cysteine-aspartic proteases; cCASP3: cleaved CASP3; CQ: chloroquine; CHX: cycloheximide; cPARP: cleaved poly (ADP-ribose) polymerase; DEPs: differential expressed proteins; ETO: etoposide; MEF: mouse embryonic fibroblast; MLKL: mixed lineage kinase domain-like; MSC: mesenchymal stem cell; MTORC1: mechanistic target of rapamycin kinase complex 1; Nec1s: necrostatin 1s; NFKB/NF-kB: nuclear factor of kappa light polypeptide gene enhancer in B cells; PLA: proximity ligation assay; RCD: regulated cell death; RIPK1: receptor (TNFRSF)-interacting serine-threonine kinase 1; RIPK3: receptor-interacting serine-threonine kinase 3; RUBCNL/PACER: RUN and cysteine rich domain containing beclin 1 interacting protein like; siCtrl: small interfering RNA nonsense; siRNA: small interfering RNA; TdT: terminal deoxynucleotidyl transferase; Tm: tunicamycin; TNF: tumor necrosis factor; TNFRSF1A/TNFR1: tumor necrosis factor receptor superfamily, member 1a.
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Previous studies have shown the relevance of high mobility group box 1 protein (HMGB1) and tumor necrosis factor α (TNFα) in nerve or tissue injury-induced nociception. However, the role of these proteins in chronic stress and social transfer of stress (STS)-induced dysfunctional pain is not entirely known. The aim of this study was to determine the participation of the spinal HMGB1-TNFα signaling pathway and TNFα receptor 1 (TNFR1) in rats subjected to chronic restraint stress (CRS) and STS. Non-stressed female and male rats in contact with CRS rats increased sniffing behavior of the anogenital area, behavior related to STS. Rats subjected to CRS and STS reduced 50 % withdrawal threshold and reached the value of tactile allodynia after 21 days of stress. Rats return to the basal withdrawal threshold after 30 days without stress and return to allodynia values in only 5 days of stress sessions (priming). Female and male rats subjected to 28 days of CRS or STS were intrathecal injected with glycyrrhizin (inhibitor of HMGB1), thalidomide (inhibitor of the TNFα synthesis), and R7050 (TNFR1 antagonist), in all the cases, an antiallodynic effect was observed. Rats under CRS or STS enhanced HMGB1 and TNFR1 protein expression in DRG and dorsal spinal cord. Data suggest that the spinal HMGB1/TNFα/TNFR1 signaling pathway plays a relevant role in the maintenance of CRS and STS-induced nociceptive hypersensitivity in rats. These proteins could be helpful in developing pain treatments for fibromyalgia in humans.
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Proteína HMGB1 , Hiperalgesia , Humanos , Ratos , Masculino , Feminino , Animais , Receptores Tipo I de Fatores de Necrose Tumoral/efeitos adversos , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteína HMGB1/efeitos adversos , Proteína HMGB1/metabolismo , Dor/induzido quimicamenteRESUMO
INTRODUCTION: Tumor necrosis factor (TNF)-α is a pro-inflammatory cytokine that promotes biomineralization in vitro in dental pulp cells. However, the role of TNF-α-TNF receptor 1 (TNFR1) signaling in reparative dentin formation and related inflammatory pathways is not known. Therefore, the aim of this study was to evaluate the role of the TNF-α-TNFR1 axis in dental pulp repair following pulp capping in vivo. METHODS: Dental pulp repair response of genetically deficient TNF-α receptor-1 mice (TNFR1-/-; n = 20) was compared with that of C57Bl6 mice (wild type [WT]; n = 20). Pulp capping was performed with mineral trioxide aggregate on the mandibular first molars of mice. After 7 and 70 days, tissues were collected and stained with hematoxylin and eosin for histopathological and histometric evaluation, and assessed by the Brown and Brenn methods for histomicrobiological analysis and by immunohistochemistry to localize TNF-α, Runt-related transcription factor 2, Dentin Sialoprotein (DSP) and Osteopontin (OPN) expression. RESULTS: Compared with WT mice, TNFR1-/- mice showed significantly decreased reparative dentin formation with a lower mineralized tissue area (P < .0001). Unlike WT mice, TNFR1-/- mice also exhibited significant dental pulp necrosis, neutrophil recruitment, and apical periodontitis formation (P < .0001) without bacterial tissue invasion. TNFR1-/- animals further exhibited decreased TNF-α, DSP, and OPN expression (P < .0001), whereas Runt-related transcription factor 2 expression was unchanged (P > .05). CONCLUSION: The TNF-α-TNFR1 axis is involved in reparative dentin formation following dental pulp capping in vivo. Genetic ablation of TNFR1 modified the inflammatory process and inhibited the expression of the DSP and OPN mineralization proteins, which culminated in dental pulp necrosis and development of apical periodontitis.
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Dentina Secundária , Periodontite Periapical , Animais , Camundongos , Hidróxido de Cálcio , Subunidade alfa 1 de Fator de Ligação ao Core , Polpa Dentária/patologia , Capeamento da Polpa Dentária/métodos , Necrose da Polpa Dentária/terapia , Necrose da Polpa Dentária/patologia , Camundongos Endogâmicos C57BL , Periodontite Periapical/patologia , Receptores Tipo I de Fatores de Necrose Tumoral , Fator de Necrose Tumoral alfaRESUMO
INTRODUCTION: The aim of this study was to investigate the role of the proinflammatory axis TNF-α-TNFR1 in experimentally induced periapical inflammation and bone resorption in mice. METHODS: After receiving Ethics Committee Approval (2019.1.139.58.0), experimental apical periodontitis was induced by means of inoculating oral microorganisms into the root canals of molars of mice. Genetically deficient tumor necrosis factor-α receptor-1 mice (TNFR1-/-; n = 50) response was compared with that of C57Bl6 wild-type mice (wild-type; n = 50) after 7, 14, 28, and 42 days. The analyses performed were micro-computed tomographic, histopathologic, histomicrobiological, and histometric evaluation, tartrate-resistant acid phosphatase staining, immunohistochemistry, and quantitative reverse transcriptase polymerase chain reaction. Data were analyzed by using one-way analysis of variance, followed by Tukey or Bonferroni tests (α = 5%). RESULTS: TNFR1-/- mice exhibited lower recruitment of neutrophils at 14, 28, and 42 days (P < .05), which resulted in reduced area and volume of apical periodontitis at 42 days (P < .05). The number of osteoclasts was also lower in TNFR1-/- animals at 14 and 42 days (P < .01), along with reduced synthesis of CTSK, MMP-9, and COX-2. Expression of RANKL, but not OPG, was reduced at 14 and 42 days (P < .001). The highest RANKL expression over OPG (ratio > 1) was found in wild-type animals at 7 (P < .0001) and 42 days (P < .001). CONCLUSIONS: Periapical inflammation and bone resorption were exacerbated in wild-type animals compared with TNFR1-/- mice, demonstrating that the TNF-α-TNFR1 signaling pathway mediated catabolic events in bone after root canal contamination.
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Reabsorção Óssea , Periodontite Periapical , Animais , Camundongos , Fator de Necrose Tumoral alfa , Receptores Tipo I de Fatores de Necrose Tumoral , Reabsorção Óssea/metabolismo , Periodontite Periapical/microbiologia , Inflamação/patologia , Transdução de Sinais , Osteoclastos/metabolismo , Ligante RANKRESUMO
OBJECTIVES: Multidrug resistance transporter 1 (Mdr-1) is a relevant component of the intestinal transcellular barrier that decreases absorption of oral drugs, thus modulating their bioavailability. Obese patients with metabolic disorders take medications that are subjected to intestinal metabolism and the Mdr-1-dependent barrier. This study evaluated the effect of a high-fat diet (HFD; 40% fat for 16 wk) on Mdr-1 expression and transport activity in C57BL/6 (C57) male mice. Comparable studies were performed in tumor necrosis factor α (TNF-α) receptor 1 knockout mice (R1KO) to delineate a possible role of TNF-α signaling. METHODS: mRNA expression was evaluated by real-time polymerase chain reaction and protein levels by western blotting and immunohistochemistry. Mdr-1 activity was assessed using the everted intestinal sac model, with rhodamine 123 as the substrate. Statistical comparisons were made using the Student t test or one-way analysis of variance followed by the post hoc Tukey test. RESULTS: Mdr-1 protein, as well as its corresponding Mdr1a and Mdr1b mRNA, was decreased in C57-HFD mice compared with controls. Immunohistochemical studies confirmed downregulation of Mdr-1 in situ. These results correlated with a 48% decrease in the basolateral to apical transport of rhodamine 123. In contrast, R1KO-HFD modified neither intestinal Mdr-1 mRNA nor its protein expression or activity. In addition, C57-HFD showed elevated intestinal TNF-α mRNA and protein (enzyme-linked immunosorbent assay) levels, whereas R1KO-HFD was undetectable or had a lower increase, respectively. CONCLUSIONS: This study demonstrated an impairment of the Mdr-1 intestinal barrier function induced by HFD as a consequence of downregulation of both Mdr-1 gene homologues, resulting in impaired Mdr-1 protein expression. Inflammatory response mediated by TNF-α receptor 1 signaling was likely involved.
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Dieta Hiperlipídica , Fator de Necrose Tumoral alfa , Camundongos , Animais , Masculino , Fator de Necrose Tumoral alfa/metabolismo , Camundongos Obesos , Rodamina 123 , Regulação para Baixo , Camundongos Endogâmicos C57BL , RNA Mensageiro , Resistência a Múltiplos MedicamentosRESUMO
Ouabain is a cardiac glycoside that has a protective effect against neuroinflammation at low doses through Na+/K+-ATPase signaling and that can activate tumor necrosis factor (TNF) in the brain. TNF plays an essential role in neuroinflammation and regulates glutamate receptors by acting on two different receptors (tumor necrosis factor receptor 1 [TNFR1] and TNFR2) that have distinct functions and expression. The activation of constitutively and ubiquitously expressed TNFR1 leads to the expression of pro-inflammatory cytokines. Thus, this study aimed to elucidate the effects of ouabain in a TNFR1 knockout (KO) mouse model. Interestingly, the hippocampus of TNFR1 KO mice showed a basal increase in both TNFR2 membrane expression and brain-derived neurotrophic factor (BDNF) release, suggesting a compensatory mechanism. Moreover, ouabain activated TNF-α-converting enzyme/a disintegrin and metalloprotease 17 (TACE/ADAM17), decreased N-methyl-D-aspartate (NMDA) receptor subunit 2A (NR2A) expression, and induced anxiety-like behavior in both genotype animals, independent of the presence of TNFR1. However, ouabain induced an increase in interleukin (IL)-1ß in the hippocampus, a decrease in IL-6 in serum, and an increase in NMDA receptor subunit 1 (NR1) only in wild-type (WT) mice, indicating that TNFR1 or TNFR2 expression may be important for some effects of ouabain. Collectively, our results indicate a connection between ouabain signaling and TNFR1, with the effect of ouabain partially dependent on TNFR1.
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BACKGROUND: Chronic inflammation due to Helicobacter pylori (H. pylori) infection promotes gastric carcinogenesis. Tumour necrosis factor-α (TNF-α), a key mediator of inflammation, induces cell survival or apoptosis by binding to two receptors (TNFR1 and TNFR2). TNFR1 can induce both survival and apoptosis, while TNFR2 results only in cell survival. The dysregulation of these processes may contribute to carcinogenesis. AIM: To evaluate the effects of TNFR1 and TNFR2 downregulation in AGS cells treated with H. pylori extract on the TNF-α pathway. METHODS: AGS cell lines containing TNFR1 and TNFR2 receptors downregulated by specific shRNAs and nonsilenced AGS cells were treated with H. pylori extract for 6 h. Subsequently, quantitative polymerase chain reaction with TaqMan® assays was used for the relative quantification of the mRNAs (TNFA, TNFR1, TNFR2, TRADD, TRAF2, CFLIP, NFKB1, NFKB2, CASP8, CASP3) and miRNAs (miR-19a, miR-34a, miR-103a, miR-130a, miR-181c) related to the TNF-α signalling pathway. Flow cytometry was employed for cell cycle analysis and apoptosis assays. RESULTS: In nonsilenced AGS cells, H. pylori extract treatment increased the expression of genes involved in cell survival and inhibited both apoptosis (NFKB1, NFKB2 and CFLIP) and the TNFR1 receptor. TNFR1 downregulation significantly decreased the expression of the TRADD and CFLIP genes, although no change was observed in the cellular process or miRNA expression. In contrast, TNFR2 downregulation decreased the expression of the TRADD and TRAF2 genes, which are both important downstream mediators of the TNFR1-mediated pathway, as well as that of the NFKB1 and CFLIP genes, while upregulating the expression of miR-19a and miR-34a. Consequently, a reduction in the number of cells in the G0/G1 phase and an increase in the number of cells in the S phase were observed, as well as the promotion of early apoptosis. CONCLUSION: Our findings mainly highlight the important role of TNFR2 in the TNF-α pathway in gastric cancer, indicating that silencing it can reduce the expression of survival and anti-apoptotic genes.
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MicroRNAs , Neoplasias Gástricas , Fator 2 Associado a Receptor de TNF/metabolismo , Apoptose , Carcinogênese , Ciclo Celular , Regulação para Baixo , Expressão Gênica , Humanos , Inflamação , MicroRNAs/genética , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/genética , Neoplasias Gástricas/genética , Proteína de Domínio de Morte Associada a Receptor de TNF/metabolismo , Fator 2 Associado a Receptor de TNF/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The study of cytokine storm in COVID-19 has been having different edges in accordance with the knowledge of the disease. Various cytokines have been the focus, especially to define specific treatments; however, there are no conclusive results that fully support any of the options proposed for emergency treatment. One of the cytokines that requires a more exhaustive review is the tumor necrosis factor (TNF) and its receptors (TNFRs) as increased values of soluble formats for both TNFR1 and TNFR2 have been identified. TNF is a versatile cytokine with different impacts at the cellular level depending on the action form (transmembrane or soluble) and the receptor to which it is associated. In that sense, the triggered mechanisms can be diversified. Furthermore, there is the possibility of the joint action provided by synergism between one or more cytokines with TNF, where the detonation of combined cellular processes has been suggested. This review aims to discuss some roles of TNF and its receptors in the pro-inflammatory stage of COVID-19, understand its ways of action, and let to reposition this cytokine or some of its receptors as therapeutic targets.
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Soluble TNF receptors (sTNFR1 and sTNFR2) are natural endogenous inhibitors of TNF and are elevated in inflammatory, autoimmune, and chronic degenerative diseases. In Chagas disease, pleiotropic cytokine TNF is considered key in immunopathology. Thus, we aimed to evaluate the levels of TNF, sTNFR1, and sTNFR2 in the serum of patients with chronic Chagas disease. TNF and its soluble receptors were quantified using Cytometric Bead Array in the serum of 132 patients, of which 51 had the indeterminate form (IND), 39 the mild cardiac form (CARD 1), 42 the severe cardiac form (CARD 2), and 20 non-infected individuals (NI). The results indicate that the soluble receptors may regulate TNF in Chagas disease, as their leves were higher in T. cruzi-infected individuals when compared to non-infected individuals. We found a moderate negative correlation between sTNFR1 and TNF in individuals with the IND form, suggesting a relationship with non-progression to more severe forms, such as heart disease. sTNFR1 and sTNFR2 were increased in all clinical forms, but with a moderate positive correlation in more severe patients (r = 0.50 and p = 0.0005). TNF levels showed no statistical differences in the groups of patients. These findings suggest the importance of the endogenous balance of the levels of soluble TNF receptors in the protection and balance in patients with chronic Chagas disease, besides revealing the immunological complexity in chronic T. cruzi-infected individuals.
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Doença de Chagas , Doença Crônica , Citocinas , Humanos , Receptores do Fator de Necrose TumoralRESUMO
The mechanism underlying the role of tumor necrosis factor alpha (TNF-α) in the development of inflammatory hyperalgesia has been extensively studied, mainly the role of TNF-α in the release of pro-inflammatory cytokines. The current concept relies in the fact that TNF-α stimulates the cascade release of other pro-inflammatory cytokines, such as IL-1ß, IL-6, and IL-8 (CINC-1 in rats), triggering the release of the final inflammatory mediator prostaglandin E2 (PGE2 ) and sympathetic amines that directly sensitize the nociceptors. However, this may not be the sole mechanism involved as the blockade of TNF-α synthesis by thalidomide prevents hyperalgesia without interrupting the synthesis of IL-1ß, IL-6, and CINC-1. Therefore, we hypothesized that activation of TNF-α receptor type 1 (TNFR1) by TNF-α increases nociceptors' susceptibility to the action of PGE2 and dopamine. We have found out that intrathecal administration of oligodeoxynucleotide-antisense (ODN-AS) against TNFR1 or thalidomide prevented carrageenan-induced hyperalgesia. The co-administration of TNF-α with a subthreshold dose of PGE2 or dopamine that does not induce hyperalgesia by itself in the hind paw of Wistar rats pretreated with dexamethasone (to prevent the endogenous release of cytokines) induced a robust hyperalgesia that was prevented by intrathecal treatment with ODN-AS against TNFR1. We consider that the activation of neuronal TNFR1 by TNF-α decisively increases the susceptibility of the peripheral afferent neuron to the action of final inflammatory mediators - PGE2 and dopamine - that ultimately induce hyperalgesia. This mechanism may also underlie the analgesic action of thalidomide.
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Receptores Tipo I de Fatores de Necrose Tumoral , Fator de Necrose Tumoral alfa , Animais , Citocinas , Hiperalgesia/induzido quimicamente , Neurônios Aferentes , Dor , Ratos , Ratos WistarRESUMO
We evaluated the role of tumor necrosis factor (TNF)-α receptor 1 (TNFR1) on ethanol-induced cardiac dysfunction. Male C57BL/6J wild-type (WT) or TNFR1-deficient mice (TNFR1-/-) were treated with ethanol (20% v/v) for 10â¯weeks. Increased protein expression of TNFR1 and NFκB p65 was detected in the left ventricle (LV) of WT mice chronically treated with ethanol. Echocardiographic analysis showed that ethanol consumption increased left ventricular posterior wall end-diastolic diameter and left ventricular posterior wall end-systolic diameter in WT, but not TNFR1-/- mice. Increased levels of TNF-α, interleukin (IL)-6, superoxide anion (O2-), thiobarbituric acid reactive substances (TBARS) as well as increased nitrotyrosine immunostaining were detected in the LV from WT, but not TNFR1-/- mice. Conversely, treatment with ethanol decreased nitrate/nitrite (NOx) concentration in the LV. Histopathological analysis showed that ethanol did not induce inflammatory infiltrates, necrosis or edema in the LV. No differences in the ventricular expression of iNOS, Nox2 or COX-2 as well as in the activity of superoxide dismutase (SOD), myeloperoxidase (MPO) and N-acetyl-beta-D-glucosaminidase (NAG) were found after treatment with ethanol. Our study provided novel evidence that ethanol consumption augmented the production of reactive oxygen species (ROS) and the synthesis of pro-inflammatory proteins in the LV through TNFR1-dependent mechanisms. These findings provided novel mechanistic insights about the contribution of TNFR1 in the initial steps of the cardiac damage induced by ethanol.
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Consumo de Bebidas Alcoólicas/metabolismo , Etanol/efeitos adversos , Mediadores da Inflamação/metabolismo , Miocárdio/metabolismo , Miocárdio/patologia , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Acetilglucosaminidase/metabolismo , Animais , Catalase/metabolismo , Doença Crônica , Citocinas/metabolismo , Eletrocardiografia , Glutationa/metabolismo , Testes de Função Cardíaca , Ventrículos do Coração/enzimologia , Ventrículos do Coração/patologia , Masculino , Camundongos Endogâmicos C57BL , Nitratos/metabolismo , Nitritos/metabolismo , Nitrosação , Estresse Oxidativo , Peroxidase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
BACKGROUND: Gastric carcinogenesis can be induced by chronic inflammation triggered by Helicobacter pylori (H. pylori) infection. Tumor necrosis factor (TNF)-α and its receptors (TNFR1 and TNFR2) regulate important cellular processes, such as apoptosis and cell survival, and the disruption of which can lead to cancer. This signaling pathway is also modulated by microRNAs (miRNAs), altering gene expression. AIM: To evaluate the mRNA and miRNAs expression involved in the TNF-α signaling pathway in gastric cancer (GC) tissues and its relationship. METHODS: Quantitative polymerase chain reaction (qPCR) by TaqMan® assay was used to quantify the RNA transcript levels of TNF-α signaling pathway (TNF, TNFR1, TNFR2, TRADD, TRAF2, CFLIP, NFKB1, NFKB2, CASP8, CASP3) and miRNAs that targets genes from this pathway (miR-19a, miR-34a, miR-103a, miR-130a, miR-181c) in 30 GC fresh tissue samples. Molecular diagnosis of H. pylori was performed by nested PCR for gene HSP60. A miRNA:mRNA interaction network was construct using Cytoscape v3.1.1 from the in silico analysis performed using public databases. RESULTS: Up-regulation of cellular survival genes as TNF, TNFR2, TRADD, TRAF2, CFLIP, and NFKB2, besides CASP8 and miR-34a was observed in GC tissues, whereas mediators of apoptosis such as TNFR1 and CASP3 were down-regulated. When the samples were stratified by histological type, the expression of miR-103a and miR-130a was significantly increased in the diffuse-type of GC compared to the intestinal-type. However, no influence of H. pylori infection was observed on the expression levels of mRNA and miRNAs analyzed. Moreover, the miRNA:mRNA interaction network showed several interrelations between the miRNAs and their target genes, highlighting miR-19a and miR-103a, which has as predicted or validated target a large number of genes in the TNF-α pathway, including TNF, TNFR1, TNFR2, CFLIP, TRADD, CASP3 and CASP8. CONCLUSION: Our findings show that cell survival genes mediated by TNF/TNFR2 binding is up-regulated in GC favoring its pro-tumoral effect, while pro-apoptotic genes as CASP3 and TNFR1 are down-regulated, indicating disbalance between apoptosis and cell proliferation processes in this neoplasm. This process can also be influenced by an intricate regulatory network of miRNA:mRNA.
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TNF-α is involved in HPV infection control by triggering cell signaling through binding in specific receptors TNFR1 and TNFR2. Genetic polymorphisms in these receptors may influence TNF-α signaling. Herein, we investigated TNFR1 rs767455 and rs2234649 single nucleotide polymorphisms, and TNFR1 protein expression in cervical squamous intraepithelial lesions (SIL) to identify their role in cervical pre-malignant development. SIL patients (n = 179) and healthy volunteers (n = 227) were enrolled for TNFR1 genotyping analysis by PCR-RFLP in blood samples and TNFR1 protein expression in cervical tissue by immunohistochemistry. No statistical differences regard genotypes and allelic frequencies for both polymorphisms were observed. Cervical TNFR1-expressing cells were rare in epithelium and basal layer regardless the groups. However, a progressive increase in infiltrating cells was observed in the stromal area, mainly in high SIL (HSIL) group compared to low SIL (LSIL, p < 0.001) and control (p < 0.001) groups. TNFR1-expressing cells frequency was higher in TNFR1 rs767455AG/GG (p < 0.001), and in rs2234649AA (p < 0.001) genotypes carries in HSIL subgroup. These data indicated that TNFR1-expression is abrogated in cervical epithelium, where HPV-induced pre-malignant lesion occurs, increasing its frequency in inflammatory cells in stroma, and is genetically controlled by TNFR1 rs767455AG/GG and rs234649AA genotypes. These biomarkers may be useful to identify cervical precancerous lesions progression.
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Early research on sepsis has focused on the initial hyper-inflammatory, cytokine mediated phase of the disorder whereas the events that govern the concomitant and subsequent anti-inflammatory compensatory response are not completely understood. In this context, the putative participation of TNFR1-mediated signaling in the immunosuppressive phase of Staphylococcus aureus sepsis has not been elucidated. The aim of this study was to determine the role of TNFR1 in directing the immune dysfunction during S. aureus sepsis and the potential contribution of MDSC to this process. Using a model of sepsis of peritoneal origin and tnfr1-/- mice, we demonstrated that during staphylococcal sepsis CD4+ T cell anergy is significantly dependent on TNFR1 expression and that signaling through this receptor has an impact on bacterial clearance in the spleen. MDSC played a major role in the generation of anergic CD4+ T cells and their accumulation in the spleen during S. aureus sepsis correlated with IL-6 induction. Although TNFR1 signaling was not required for MDSC accumulation and expansion in the spleen, it determined the in vivo expression of Arginase 1 and iNOS, enzymes known to participate in the suppressive function of this population. Moreover, our data indicate that TNFR1-mediated IL-10 production may modulate MDSC function during staphylococcal sepsis. Taken together these results indicate that TNFR1 plays a critical role on T cell dysfunction during S. aureus sepsis by regulating immunomodulatory mediators in MDSC. The role of TNFR1-mediated signaling during the immunosuppressive phase of staphylococcal sepsis should be considered when designing novel alternative therapeutic approaches.
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Linfócitos T CD4-Positivos/imunologia , Terapia de Imunossupressão , Células Supressoras Mieloides/imunologia , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Sepse/patologia , Transdução de Sinais , Infecções Estafilocócicas/patologia , Animais , Arginase/metabolismo , Interleucina-6/metabolismo , Camundongos , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/deficiência , Sepse/imunologia , Baço/patologia , Infecções Estafilocócicas/imunologiaRESUMO
Obesity is accompanied by a low-grade inflammation state, characterized by increased proinflammatory cytokines levels such as tumor necrosis factor alpha (TNFα) and interleukin-1 beta (IL-1ß). In this regard, there exists a lack of studies in hepatic tissue about the role of TNFα receptor 1 (TNFR1) in the context of obesity and insulin resistance during the progression of nonalcoholic fatty liver disease (NAFLD). The aim of this work was to evaluate the effects of high-caloric feeding (HFD) (40% fat, for 16 weeks) on liver inflammation-induced apoptosis, insulin resistance, hepatic lipid accumulation and its progression toward nonalcoholic steatohepatitis (NASH) in TNFR1 knock-out and wild-type mice. Mechanisms involved in HFD-derived IL-1ß release and impairment of insulin signaling are still unknown, so we determined whether IL-1ß affects liver insulin sensitivity and apoptosis through TNFα receptor 1 (TNFR1)-dependent pathways. We showed that knocking out TNFR1 induces an enhanced IL-1ß plasmatic release upon HFD feed. This was correlated with higher hepatic and epididymal white adipose tissue mRNA levels. In vivo and in vitro assays confirmed an impairment in hepatic insulin signaling, in part due to IL-1ß-induced decrease of AKT activation and diminution of IRS1 levels, followed by an increase in inflammation, macrophage (resident and recruited) accumulation, hepatocyte apoptotic process and finally hepatic damage. In addition, TNFR1 KO mice displayed higher levels of pro-fibrogenic markers. TNFR1 signaling disruption upon an HFD leads to an accelerated progression from simple steatosis to a more severe phenotype with many NASH features, pointing out a key role of TNFR1 in NAFLD progression.
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Dieta Hiperlipídica/efeitos adversos , Hepatopatia Gordurosa não Alcoólica/etiologia , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Animais , Apoptose/genética , Insulina/metabolismo , Resistência à Insulina , Interleucina-1beta/metabolismo , Fígado/metabolismo , Fígado/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/patologia , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Transdução de SinaisRESUMO
BACKGROUND: In conditions of immunosuppression, the central nervous sty 5ystem (CNS) is the main target tissue for the reactivation of infection by Trypanosoma cruzi, the causative agent of Chagas disease. In experimental T. cruzi infection, interferon gamma (IFNγ)+ microglial cells surround astrocytes harboring amastigote parasites. In vitro, IFNγ fuels astrocyte infection by T. cruzi, and IFNγ-stimulated infected astrocytes are implicated as potential sources of tumor necrosis factor (TNF). Pro-inflammatory cytokines trigger behavioral alterations. In T. cruzi-infected mice, administration of anti-TNF antibody hampers depressive-like behavior. Herein, we investigated the effects of TNF on astrocyte susceptibility to T. cruzi infection and the regulation of cytokine production. METHODS: Primary astrocyte cultures of neonatal C57BL/6 and C3H/He mice and the human U-87 MG astrocyte lineage were infected with the Colombian T. cruzi strain. Cytokine production, particularly TNF, and TNF receptor 1 (TNFR1/p55) expression were analyzed. Recombinant cytokines (rIFNγ and rTNF), the anti-TNF antibody infliximab, and the TNFR1 modulator pentoxifylline were used to assess the in vitro effects of TNF on astrocyte susceptibility to T. cruzi infection. To investigate the role of TNF on CNS colonization by T. cruzi, infected mice were submitted to anti-TNF therapy. RESULTS: rTNF priming of mouse and human astrocytes enhanced parasite/astrocyte interaction (i.e., the percentage of astrocytes invaded by trypomastigote parasites and the number of intracellular parasite forms/astrocyte). Furthermore, T. cruzi infection drove astrocytes to a pro-inflammatory profile with TNF and interleukin-6 production, which was amplified by rTNF treatment. Adding rTNF prior to infection fueled parasite growth and trypomastigote egression, in parallel with increased TNFR1 expression. Importantly, pentoxifylline inhibited the TNF-induced increase in astrocyte susceptibility to T. cruzi invasion. In T. cruzi-infected mice, anti-TNF therapy reduced the number of amastigote nests in the brain. CONCLUSIONS: Our data implicate TNF as a promoter of T. cruzi invasion of mouse and human astrocytes. Moreover, the TNF-enriched inflammatory milieu and enhanced TNFR1 expression may favor TNF signaling, astrocyte colonization by T. cruzi and egression of trypomastigotes. Therefore, in T. cruzi infection, a self-sustaining TNF-induced inflammatory circuit may perpetuate the parasite cycle in the CNS and ultimately promote cytokine-driven behavioral alterations.
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Astrócitos/metabolismo , Doença de Chagas/metabolismo , Mediadores da Inflamação/metabolismo , Trypanosoma cruzi , Fator de Necrose Tumoral alfa/toxicidade , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Linhagem Celular Tumoral , Células Cultivadas , Doença de Chagas/patologia , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Humanos , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: TNF-α is a key cytokine involved in prostate carcinogenesis and is mediated by the TNF-α receptor type 1 (TNFR-1). This receptor triggers two opposite pathways: cell death or cell survival and presents a protective or stimulator role in cancer. Thus, the purpose of this study was to evaluate the role of TNF signaling in chemically induced prostate carcinogenesis in mice. METHODS: C57bl/6 wild type (WT) and p55 TNFR-1 knockout mice (KO) were treated with mineral oil (control) or N-methyl N-nitrosurea (MNU) in association with testosterone (MNU+T, single injection of 40 mg/kg and weekly injection 2 mg/kg, respectively) over the course of 6 months. After this induction period, prostate samples were processed for histological and biochemical analysis. RESULTS: MNU+T treatment led to the development of prostate intraepithelial neoplasia (PIN) and adenocarcinoma (PCa) in both WT and KO animals; however, the incidence of PCa was lower in KO group than in WT. Cell proliferation analysis showed that PCNA levels were significantly lower in the KO group, even after carcinogenesis induction. Furthermore, the prostate of KO animals had lower levels of p65 and p-mTOR after treatment with MNU+T than WT. There was also a decrease in prostate androgen receptor levels after induction of carcinogenesis in both KO and WT mice. Regarding the extracellular matrix in the prostate, KO mice had higher levels of fibronectin and lower levels of matrix metalloproteinase 2 (MMP2) after carcinogenesis. Finally, there was a similar increase in apoptosis in both groups after carcinogenesis, indicating that the TNAFr1 pathway in prostate carcinogenesis presented proliferative, and not apoptotic, stimuli. CONCLUSIONS: TNF-α, through its receptor TNFR-1, promoted cell proliferation and cell survival in prostate by activation of the AKT/mTOR and NFKB pathway, which stimulated prostate carcinogenesis in chemically induced mice. Prostate 76: 917-926, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Carcinogênese , Neoplasias da Próstata , Receptores Tipo I de Fatores de Necrose Tumoral/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Adenocarcinoma/patologia , Animais , Apoptose , Carcinogênese/patologia , Proliferação de Células , Sobrevivência Celular , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Antígeno Nuclear de Célula em Proliferação/análise , Neoplasia Prostática Intraepitelial/patologia , Neoplasias da Próstata/química , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Androgênicos/análise , Receptores Tipo I de Fatores de Necrose Tumoral/deficiência , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Serina-Treonina Quinases TOR/análise , Serina-Treonina Quinases TOR/metabolismo , Fator de Transcrição RelA/análiseRESUMO
In Chagas disease, understanding how the immune response controls parasite growth but also leads to heart damage may provide insight into the design of new therapeutic strategies. Tumor necrosis factor-alpha (TNF-á) is important for resistance to acute Trypanosoma cruzi infection; however, in patients suffering from chronic T. cruzi infection, plasma TNF-á levels correlate with cardiomyopathy. Recent data suggest that CD8-enriched chagasic myocarditis formation involves CCR1/CCR5-mediated cell migration. Herein, the contribution of TNF-á, especially signaling through the receptor TNFR1/p55, to the pathophysiology of T. cruzi infection was evaluated with a focus on the development of myocarditis and heart dysfunction. Colombian strain-infected C57BL/6 mice had increased frequencies of TNFR1/p55+ and TNF-á+ splenocytes. Although TNFR1-/- mice exhibited reduced myocarditis in the absence of parasite burden, they succumbed to acute infection. Similar to C57BL/6 mice, Benznidazole-treated TNFR1-/- mice survived acute infection. In TNFR1-/- mice, reduced CD8-enriched myocarditis was associated with defective activation of CD44+CD62Llow/- and CCR5+ CD8+ lymphocytes. Also, anti-TNF-á treatment reduced the frequency of CD8+CCR5+ circulating cells and myocarditis, though parasite load was unaltered in infected C3H/HeJ mice. TNFR1-/- and anti-TNF-á-treated infected mice showed regular expression of connexin-43 and reduced fibronectin deposition, respectively. Furthermore, anti-TNF-á treatment resulted in lower levels of CK-MB, a cardiomyocyte lesion marker. Our results suggest that TNF/TNFR1 signaling promotes CD8-enriched myocarditis formation and heart tissue damage, implicating the TNF/TNFR1 signaling pathway as a potential therapeutic target for control of T. cruzi-elicited cardiomyopathy.