Angiotensin II-induced natriuresis is attenuated in knockout mice lacking the receptors for tumor necrosis factor-α.

Intravenous infusion of relatively higher doses of angiotensin II (AngII) elicits natriuresis as opposed to its usual anti-natruretic response. As AngII can induce tumor necrosis factor-α (TNFα) production which elicits natriuresis via its action on TNFα receptor type 1 (TNFR1), we hypothesize that the concomitant release of TNFα contributes to the natriuretic response to AngII. Responses to AngII infusion (1 ng min-1  g-1 for 75 min, iv) were evaluated in anesthetized knockout (KO) mice lacking TNFR1 (n = 6) and TNFR2 (TNFα receptor type 2; n = 6) and compared these responses with those in wild type (WT; n = 6) mice. Arterial pressure (AP) was recorded from a cannula placed in the carotid artery. Renal blood flow (RBF) and glomerular filtration rate (GFR) were measured by PAH and inulin clearances, respectively. Urine was collected from a catheter placed in the bladder. AngII caused similar increases (p < 0.05 vs basal values) in AP (WT, 37 ± 5%; TNFR1KO, 35 ± 4%; TNFR2KO, 30 ± 4%) and decreases (p < 0.05) in RBF (WT, -39 ± 5%; TNFR1KO, -28 ± 6%; TNFR2KO, -31 ± 4%) without significant changes in GFR (WT, -17 ± 7%; TNFR1KO, -18 ± 7%; TNFR2KO, -12 ± 7%). However, despite similar changes in AP and renal hemodynamics, AngII induced increases (p < 0.05) in urinary sodium excretion in WT (3916 ± 942%) were less in the KO strains, more or less in TNFR1KO (473 ± 170%) than in TNFR2KO (1176 ± 168%). These data indicate that TNF-α receptors, particularly TNFR1 are involved in the natriuretic response that occur during acute infusion of AngII and thus, plays a protective role in preventing excessive salt retention at clinical conditions associated with elevated AngII level.

Overexpression of TNFα induces senescence, autophagy and mitochondrial dysfunctions in melanoma cells.

BACKGROUND: Tumor necrosis factor alpha (TNFα) is a pleiotropic cytokine with both anti-tumorigenic and pro-tumorigenic activity, affecting tumor cell biology, the balance between cell survival and death. The final effect of TNFα is dependent on the type of malignant cells, with the potential to arrest cancer progression. METHODS: In order to explain the diverse cellular response to TNFα, we engineered melanoma and colorectal carcinoma cell lines stably overexpressing this cytokine. RESULTS: Under the TNFα overexpression, significant upregulation of two genes was observed: proinflammatory cytokine IL6 gene in melanoma cells A375 and gene for pro-apoptotic ligand TRAIL in colorectal carcinoma cells HT29, both mediated by TNFα/TNFR1 signaling. Malignant melanoma line A375 displayed also increased autophagy on day 3, followed by premature senescence on day 6. Both processes seem to be interconnected, following earlier apoptosis induction and deregulation of mitochondrial functions. We documented altered mitochondrial status, lowered ATP production, lowered mitochondrial mass, and changes in mitochondrial morphology (shortened and condensed mitochondria) both in melanoma and colorectal carcinoma cells. Overexpression of TNFα was not linked with significant affection of the subpopulation of cancer stem-like cells in vitro. However, we could demonstrate a decrease in aldehyde dehydrogenase (ALDH) activity up to 50%, which is associated with to the stemness phenotype. CONCLUSIONS: Our in vitro study of direct TNFα influence demonstrates two distinct outcomes in tumor cells of different origin, in non-epithelial malignant melanoma cells of neural crest origin, and in colorectal carcinoma cells derived from the epithelium.

High-glucose concentration aggravates TNF-alpha-induced cell viability reduction in human CD146-positive periodontal ligament cells via TNFR-1 gene demethylation.

Periodontitis is a chronic inflammatory disease that results in the destruction of periodontal soft tissue and the resorption of alveolar bone. Evidence indicates that in diabetic patients, hyperglycemia suppresses periodontal ligament stem cell (PDLSC) functions and leads to difficulties in periodontal repair. The present study aimed to explore the mechanisms by which high-glucose concentrations aggravate cell viability reduction in human CD146-positive PDLCs (CD146+ PDLCs) under tumor necrosis factor-alpha (TNF-alpha) induction. CD146+ PDLCs were isolated from periodontal ligament tissues and treated in the absence or presence of 10 ng/ml of TNF-alpha and 30 mM glucose. Cell viability was detected using Cell Counting Kit-8 assays and Luminescent Cell Viability Assays. Western blotting and real-time polymerase chain reaction were performed to determine tumor necrosis factor-alpha receptor-1 (TNFR-1) protein and messenger RNA expression. Bisulfite and MassArray methylation analyses were used to analyze the methylation status of the TNFR-1 gene. Our results indicated that cell viability was reduced after treatment with a combination of both high-glucose concentration and TNF-alpha. Treatment with 30 mM glucose suppressed DNA methyltransferase (DNMT) activities and DNMT1 protein expression, and this was accompanied by the upregulation of TNFR-1. Additionally, we found that the CpG island located within the TNFR-1 gene was hypomethylated under 30 mM glucose conditions. S-adenosylmethionine, an established methyl donor, reversed TNFR-1 upregulation and restored cell viability against high-glucose concentration and TNF-alpha. In conclusion, the present findings suggest that high-glucose-induced CpG island hypomethylation within the TNFR-1 gene plays an essential role in TNFR-1 upregulation, and this further enhances the cell viability reduction of CD146+ PDLCs caused by TNF-alpha.

Autoantibodies from SLE patients induce programmed cell death in murine fibroblast cells through interaction with TNFR1 receptor.

Various pathological processes are known to be associated with the production of IgG autoantibodies, which have high affinity for self-antigens and often cause tissue injury and the development of autoimmune diseases. However, the mechanism of their cytotoxic activity is not clearly understood yet. Here, we have shown that the action of these autoantibodies on cells expressing TNFR1 (the cell surface receptor for TNFα) can cause both caspase-dependent apoptosis and necroptosis of these cells, with suppression of apoptosis resulting in switching to RIP1-dependent necroptosis. Analysis of necroptotic mechanisms has shown that a critical point of necroptosis is phosphorylation of RIP1 and RIP3 kinases, which is followed by the involvement of lysosomes and mitochondria in this process. The induction of cytotoxicity is initiated by the interaction of autoantibodies with TNFR1, and autoantibodies can therefore be regarded as a new functional ligand for this receptor. The innate immunity protein Tag7 (PGLYRP1) described in our recent studies is also a ligand for TNFR1 and competes with autoantibodies for binding with it. Supposedly, the cytotoxic effect of autoantibodies is one of the factors responsible for autoimmune diseases that lead to tissue injury.

Ninjurin-1 upregulated by TNFα receptor 1 stimulates monocyte adhesion to human TNFα-activated endothelial cells; benefic effects of amlodipine.

AIMS: The objectives of the present study were to investigate the mechanisms of Ninj-1 regulation in TNFα-activated human endothelial cells (HEC), and to test if Amlodipine (AML) ameliorates the inflammatory stress by decreasing Ninj-1 expression. MAIN METHODS: TNFα-activated HEC with/without AML (0.1 µM and 1 µM) were used. TNFα-receptor 1 (TNFR1) was silenced and inhibitors for oxidative stress (N-acetyl cysteine), endoplasmic reticulum stress (salubrinal, 4-phenyl butyric acid), or NF-kB (Bay 11-7085) and p38 MAPK (SB203580) were used. Levels of Ninj-1, TNFR1, monocyte adhesion, endoplasmic reticulum stress (ERS) sensors, NADPH oxidase- and mitochondria-derived oxidative species were evaluated. KEY FINDINGS: The novel findings that we report here are: (i) silencing the endothelial TNFR1 leads to decreased Ninj-1 expression and diminished monocyte adhesion; (ii) increased oxidative stress, ERS and NF-kB activation enhance Ninj-1 expression and monocyte adhesion; (iii) up-regulation of endothelial Ninj-1 expression stimulates monocytes adhesion to TNFα - activated HEC; (iv) AML diminishes monocyte adhesion by reducing Ninj-1 expression through mechanisms involving the decrease of NADPH oxidase and mitochondria-dependent oxidative stress, ERS and NF-kB. In addition, AML alleviates apoptosis by reducing the pro-apoptotic CHOP expression and re-establishing the mitochondrial transmembrane potential. SIGNIFICANCE: The results of the present study suggest that Ninj-1 and the proteins involved in its regulation can be considered therapeutic targets for the alleviation of inflammation- dependent disorders. In addition, we demonstrate that some of the benefic effects of AML can be achieved through regulation of Ninj-1.

Injury Pathways That Lead to AKI in a Mouse Kidney Transplant Model.

BACKGROUND: Prolonged cold ischemia (CI) is a risk factor for acute kidney injury after kidney transplantation. We endeavored to determine the pathways involved in the development of tubular cell injury and death before and after transplantation. We hypothesized that ex vivo cold storage before transplant would produce a different injury phenotype to that seen after engraftment in kidney transplants with or without CI. METHODS: Four groups of mouse donor kidneys were studied: (1) nontransplanted control kidneys; (2) donor kidneys subjected to ex vivo cold ischemia (CI); (3) donor kidneys subjected to kidney transplant without CI (Txp); and (4) donor kidneys subjected to CI followed by transplantation (CI+Txp). RESULTS: Acute kidney injury only occurred in the CI+Txp group, which had significantly increased sCr versus the Txp group and the control mice. Histologically, the CI group demonstrated significantly increased tubular cell apoptosis and caspase-9 expression, whereas the Txp group demonstrated only mild brush border injury without apoptosis or necrosis. In contrast, the CI+Txp group had tubular cell apoptosis associated with expression of caspase-8, TNFR1, and increased serum TNF-α. CI+Txp also led to significantly higher ATN scores in association with increased RIP1, RIP3, pMLKL, and TLR4 expression. CONCLUSIONS: Our results suggest distinct therapies are needed at different times during organ preservation and transplantation. Prevention of apoptosis during cold storage is best achieved by inhibiting intrinsic pathways. In contrast, prevention of cell death and innate immunity after CI+Txp requires inhibition of both the extrinsic death receptor pathway via TNFR1 and caspase-8 and inhibition of programmed necrosis via TLR4 and TNFR1.

Morphine withdrawal recruits lateral habenula cytokine signaling to reduce synaptic excitation and sociability.

The lateral habenula encodes aversive stimuli contributing to negative emotional states during drug withdrawal. Here we report that morphine withdrawal in mice leads to microglia adaptations and diminishes glutamatergic transmission onto raphe-projecting lateral habenula neurons. Chemogenetic inhibition of this circuit promotes morphine withdrawal-like social deficits. Morphine withdrawal-driven synaptic plasticity and reduced sociability require tumor necrosis factor-α (TNF-α) release and neuronal TNF receptor 1 activation. Hence, habenular cytokines control synaptic and behavioral adaptations during drug withdrawal.

A protein-protein interaction map of the TNF-induced NF-κB signal transduction pathway.

Tumor Necrosis Factor (TNF) has a crucial role in inflammation, cell proliferation and cell death. Dysregulation of TNF receptor 1 (TNFR1)-induced Nuclear Factor-kappa B (NF-κB) signaling leads to chronic inflammation and is associated with several human inflammatory pathologies. Hence, TNF neutralization suppresses inflammation and attenuates inflammatory pathology. However, despite its beneficial effects, anti-TNF therapy suffers from efficacy issues and severe immune side effects. There is thus an urging need to identify novel targets for pharmaceutical intervention in the NF-κB signaling pathway. Here, we present a protein-protein interaction dataset of the TNFR1-induced signaling pathway. For this, we used Virotrap, a novel method for studying protein complexes without disrupting the cellular integrity, on 12 central proteins controlling NF-κB and cell death signaling, both under resting conditions as well as upon TNF stimulation. Our dataset reveals dynamic interactions in TNFR1-induced NF-κB signaling and identifies both known as well as novel interactors that may help to further unravel the molecular mechanisms steering TNF-induced inflammatory signaling and pathology.

S-Nitrosylation of cIAP1 Switches Cancer Cell Fate from TNFα/TNFR1-Mediated Cell Survival to Cell Death.

TNFα is a prominent proinflammatory cytokine and a critical mediator for the development of many types of cancer such as breast, colon, prostate, cervical, skin, liver, and chronic lymphocytic leukemia. Binding of TNFα to TNFR1 can lead to divergent signaling pathways promoting predominantly NF-κB activation but also cell death. We report here that the nitric oxide (NO) donor glyceryl trinitrate (GTN) converts TNFα, generated from immune cells or cancer cells stimulated by chemotherapy, into a prodeath mediator in colon and mammary cancer cells. GTN-mediated S-nitrosylation of cIAP1 on cysteines 571 and 574 inhibited its E3 ubiquitin ligase activity, which in turn reduced Lys63-linked ubiquitination of RIP1 and initiated assembly of a death complex. These findings provide insights into how NO can harness advantageous aspects of inflammation in cancer and provide new therapeutic strategies.Significance: Combination of an NO donor with chemotherapeutic drug-induced TNFα represents a potentially valuable anticancer strategy. Cancer Res; 78(8); 1948-57. ©2018 AACR.

Leptin Mediates In Vivo Neutrophil Migration: Involvement of Tumor Necrosis Factor-Alpha and CXCL1.

Leptin directly activates macrophages and lymphocytes, but the role of leptin in neutrophil activation and migration is still controversial. Here, we investigate the in vivo mechanisms of neutrophil migration induced by leptin. The intraperitoneal injection of leptin (1 mg/kg) induces a time- and concentration-dependent neutrophil influx. We did not observe the enhancement of lipid bodies/droplets in neutrophils, after leptin treatment, as we had observed previously in peritoneal macrophages. The participation of leukotriene B4 (LTB4) in neutrophil recruitment triggered by leptin was investigated using different strategies. Leptin-induced neutrophil recruitment occurs both in the absence of 5-lipoxygenase activity in 5-lipoxygenase (5-LO)-/- mice and after the administration of either 5-LO inhibitor (Zileuton) or the LTB4 receptor antagonist (U-75302). Moreover, no direct induction of LTB4 by leptin could be observed. Neutrophil influx could not be prevented by the mammalian target of rapamycin (mTOR) inhibitor, rapamycin, contrasting with the leptin-induced signaling for lipid body formation in macrophage that is mTOR-dependent. Leptin administration led to tumor necrosis factor-alpha (TNFα) production by the peritoneal cells both in vivo and in vitro. In addition, neutrophil recruitment was inhibited in tumor necrosis factor receptor 1 (TNFR1-/-) mice, indicating a role for TNF in leptin-induced neutrophil recruitment to the peritoneal cavity. Leptin-induced neutrophil influx was PI3Kγ-dependent, as it was absent in PI3Kγ-/- mice. Accordingly, leptin induced the peritoneal cells to produce CXCL1, both in vivo and in vitro, and the neutrophil influx was ablated after using an antibody against CXCL1. Our results establish TNFα/TNFR1- and CXCL1-dependent signaling as important pathways for leptin-induced neutrophil migration in vivo.

Simultaneous Inhibition of Tumor Necrosis Factor Receptor 1 and Matrix Metalloproteinase 8 Completely Protects Against Acute Inflammation and Sepsis.

OBJECTIVES: Sepsis causes very high mortality and morbidity rates and remains one of the biggest medical challenges. This study investigates whether plasma levels of both matrix metalloproteinase 8 and tumor necrosis factor receptor 1 are associated with sepsis severity and also investigates the therapeutic applicability of simultaneous inhibition of the two molecules in sepsis. DESIGN: Observational human pilot study-prospective controlled animal study. SETTING: University hospital and research laboratory. SUBJECTS: Sepsis patients and C57BL/6 mice deficient for matrix metalloproteinase 8 and/or tumor necrosis factor receptor 1. INTERVENTION: Plasma and whole blood RNA were collected from 13 sepsis patients for 7 consecutive days and within 24 hours of admission to ICU. Matrix metalloproteinase 8 and tumor necrosis factor receptor 1 plasma and expression levels were determined in these patients. Mice deficient for both matrix metalloproteinase 8 and tumor necrosis factor receptor 1 were generated and subjected to endotoxemia and cecal ligation and puncture. Additionally, a bispecific Nanobody that simultaneously blocks matrix metalloproteinase 8 and tumor necrosis factor receptor 1 was created. MEASUREMENTS AND MAIN RESULTS: Plasma levels of matrix metalloproteinase 8 and tumor necrosis factor receptor 1 were positively correlated with the Sequential Organ Failure Assessment score (r, 0.51 and 0.58) and interleukin 6 levels (r, 0.59 and 0.52) in 13 sepsis patients. Combined elimination of tumor necrosis factor receptor 1 and matrix metalloproteinase 8 in double knockout mice resulted in superior survival in endotoxemia and CLP compared with single knockouts and wild-type mice. Cotreatment with our bispecific Nanobody in CLP resulted in improved survival rates (28% vs 19%) compared with untreated mice. CONCLUSIONS: Inhibition of matrix metalloproteinase 8 and tumor necrosis factor receptor 1 might have therapeutic potential to treat sepsis and proof-of-principle was provided as therapeutics that inhibit both tumor necrosis factor receptor 1 and matrix metalloproteinase 8 are effective in CLP.

Reduced internalization of TNF-ɑ/TNFR1 down-regulates caspase dependent phagocytosis induced cell death (PICD) in neonatal monocytes.

Phagocytosis-induced cell death (PICD) is diminished in cord blood monocytes (CBMO) as compared to cells from adults (PBMO) due to differences in the CD95-pathway. This may support a prolonged pro-inflammatory response with sequels of sustained inflammation as seen in neonatal sepsis. Here we hypothesized that TNF-α mediated induction of apoptosis is impaired in CBMO due to differences in the TNFR1-dependent internalization. Monocytes were infected with Escherichia coli-GFP (E. coli-GFP). Monocyte phenotype, phagocytic activity, induction of apoptosis, and TNF-α/TNF-receptor (TNFR) -expression were analysed. In the course of infection TNF-α-secretion of CBMO was reduced to 40% as compared to PBMO (p<0.05). Neutralization of TNF-α by an αTNF-α antibody reduced apoptotic PICD in PBMO four-fold (p < 0.05 vs. infection with E. coli). PICD in CBMO was reduced 5-fold compared to PBMO and showed less responsiveness to αTNF-α antibody. CBMO expressed less pro-apoptotic TNFR1, which, after administration of TNF-α or infection with E. coli was internalized to a lesser extent. With similar phagocytic capacity, reduced TNFR1 internalization in CBMO was accompanied by lower activation of caspase-8 (p < 0.05 vs. PBMO). Stronger caspase-8 activation in PBMO caused more activation of effector caspase-3 and apoptosis (all p < 0.05 vs. PBMO). Our results demonstrate that TNFR1 internalization is critical in mediating PICD in monocytes after infection with E.coli and is reduced in CBMO.

Regulation of RIPK1 activation by TAK1-mediated phosphorylation dictates apoptosis and necroptosis.

Stimulation of TNFR1 by TNFα can promote three distinct alternative mechanisms of cell death: necroptosis, RIPK1-independent and -dependent apoptosis. How cells decide which way to die is unclear. Here, we report that TNFα-induced phosphorylation of RIPK1 in the intermediate domain by TAK1 plays a key role in regulating this critical decision. Using phospho-Ser321 as a marker, we show that the transient phosphorylation of RIPK1 intermediate domain induced by TNFα leads to RIPK1-independent apoptosis when NF-κB activation is inhibited by cycloheximide. On the other hand, blocking Ser321 phosphorylation promotes RIPK1 activation and its interaction with FADD to mediate RIPK1-dependent apoptosis (RDA). Finally, sustained phosphorylation of RIPK1 intermediate domain at multiple sites by TAK1 promotes its interaction with RIPK3 and necroptosis. Thus, absent, transient and sustained levels of TAK1-mediated RIPK1 phosphorylation may represent distinct states in TNF-RSC to dictate the activation of three alternative cell death mechanisms, RDA, RIPK1-independent apoptosis and necroptosis.TNFα can promote three distinct mechanisms of cell death: necroptosis, RIPK1-independent and dependent apoptosis. Here the authors show that TNFα-induced phosphorylation of RIPK1 in the intermediate domain by TAK1 plays a key role in regulating this decision.

Death of adrenocortical cells during murine acute T. cruzi infection is not associated with TNF-R1 signaling but mostly with the type II pathway of Fas-mediated apoptosis.

Earlier studies from our laboratory demonstrated that acute experimental Trypanosoma cruzi infection promotes an intense inflammation along with a sepsis-like dysregulated adrenal response characterized by normal levels of ACTH with raised glucocorticoid secretion. Inflammation was also known to result in adrenal cell apoptosis, which in turn may influence HPA axis uncoupling. To explore factors and pathways which may be involved in the apoptosis of adrenal cells, together with its impact on the functionality of the gland, we carried out a series of studies in mice lacking death receptors, such as TNF-R1 (C57BL/6-Tnfrsf1a tm1Imx or TNF-R1-/-) or Fas ligand (C57BL/6 Fas-deficient lpr mice), undergoing acute T. cruzi infection. Here we demonstrate that the late hypercorticosterolism seen in C57BL/6 mice during acute T. cruzi infection coexists with and hyperplasia and hypertrophy of zona fasciculata, paralleled by increased number of apoptotic cells. Apoptosis seems to be mediated mainly by the type II pathway of Fas-mediated apoptosis, which engages the mitochondrial pathway of apoptosis triggering the cytochrome c release to increase caspase-3 activation. Fas-induced apoptosis of adrenocortical cells is also related with an exacerbated production of intra-adrenal cytokines that probably maintain the late supply of adrenal hormones during host response. Present results shed light on the molecular mechanisms dealing with these phenomena which are crucial not only for the development of interventions attempting to avoid adrenal dysfunction, but also for its wide occurrence in other infectious-based critical illnesses.

Autonomous TNF is critical for in vivo monocyte survival in steady state and inflammation.

Monocytes are circulating mononuclear phagocytes, poised to extravasate to sites of inflammation and differentiate into monocyte-derived macrophages and dendritic cells. Tumor necrosis factor (TNF) and its receptors are up-regulated during monopoiesis and expressed by circulating monocytes, as well as effector monocytes infiltrating certain sites of inflammation, such as the spinal cord, during experimental autoimmune encephalomyelitis (EAE). In this study, using competitive in vitro and in vivo assays, we show that monocytes deficient for TNF or TNF receptors are outcompeted by their wild-type counterpart. Moreover, monocyte-autonomous TNF is critical for the function of these cells, as TNF ablation in monocytes/macrophages, but not in microglia, delayed the onset of EAE in challenged animals and was associated with reduced acute spinal cord infiltration of Ly6Chi effector monocytes. Collectively, our data reveal a previously unappreciated critical cell-autonomous role of TNF on monocytes for their survival, maintenance, and function.

TNFR1 and TNFR2 differentially mediate TNF-α-induced inflammatory responses in rheumatoid arthritis fibroblast-like synoviocytes.

TNF-α has long been implicated in the progression of rheumatoid arthritis (RA). However, how the receptors of TNF-α, namely TNFR1 and TNFR2, mediate TNF-α-induced inflammatory responses in fibroblast-like synoviocytes (FLS) in RA has not been elucidated. In the present study, primary FLS cells were isolated from RA patients and treated with TNF-α in vitro. The exogenous TNF-α induced the expression and release of endogenous TNF-α in FLS. In addition, TNF-α led to gradual downregulation of TNFR1 following 1 h treatment. By contrast, the expression of TNFR2 was markedly upregulated after 12 h treatment with TNF-α. Moreover, following TNF-α treatment, the expression of interleukin (IL)-2, IL-6, and IL-8 was gradually increased with time, but their mRNA levels dropped significantly at 48 h. We further investigated the differential functions of TNFR1 and TNFR2 in FLS by conducting siRNA-mediated knockdown. The TNF-α autocrine was inhibited to a greater extent in TNFR1-silenced FLS compared with TNFR2-silenced FLS. Silencing of TNFR1, not TNFR2, activated intrinsic apoptosis and inhibited TNF-α-induced cytokine production in FLS. These results suggest that TNFR1 is the major pro-inflammatory mediator of TNF-α in FLS, whereas TNFR2, which is upregulated in response to prolonged TNF-α stimulation, may act as an immunosuppressor in FLS for the prevention of overwhelming inflammatory reactions.

Hyperoxaluria Requires TNF Receptors to Initiate Crystal Adhesion and Kidney Stone Disease.

Intrarenal crystals trigger inflammation and renal cell necroptosis, processes that involve TNF receptor (TNFR) signaling. Here, we tested the hypothesis that TNFRs also have a direct role in tubular crystal deposition and progression of hyperoxaluria-related CKD. Immunohistochemical analysis revealed upregulated tubular expression of TNFR1 and TNFR2 in human and murine kidneys with calcium oxalate (CaOx) nephrocalcinosis-related CKD compared with controls. Western blot and mRNA expression analyses in mice yielded consistent data. When fed an oxalate-rich diet, wild-type mice developed progressive CKD, whereas Tnfr1-, Tnfr2-, and Tnfr1/2-deficient mice did not. Despite identical levels of hyperoxaluria, Tnfr1-, Tnfr2-, and Tnfr1/2-deficient mice also lacked the intrarenal CaOx deposition and tubular damage observed in wild-type mice. Inhibition of TNFR signaling prevented the induced expression of the crystal adhesion molecules, CD44 and annexin II, in tubular epithelial cells in vitro and in vivo, and treatment with the small molecule TNFR inhibitor R-7050 partially protected hyperoxaluric mice from nephrocalcinosis and CKD. We conclude that TNFR signaling is essential for CaOx crystal adhesion to the luminal membrane of renal tubules as a fundamental initiating mechanism of oxalate nephropathy. Furthermore, therapeutic blockade of TNFR might delay progressive forms of nephrocalcinosis in oxalate nephropathy, such as primary hyperoxaluria.

Role of the type I tumor necrosis factor receptor in inflammation-associated olfactory dysfunction.

BACKGROUND: To understand mechanisms of human olfactory dysfunction in chronic rhinosinusitis, an inducible olfactory inflammation (IOI) model has been utilized to chronically express inflammatory cytokines locally, resulting in neuronal loss, diminished odorant responses, and repressed olfactory regeneration. Knockout of the minor tumor necrosis factor α receptor 2 (TNFR2) was previously shown to partially rescue these olfactory changes. The purpose of current study was to investigate the role of the major TNF receptor, TNFR1, in chronic olfactory inflammation. METHODS: Two experimental groups of mice were studied: TNFR1 knockout in IOI background and TNFR1 knockout with allergen-induced inflammation. Olfactory function was assayed by electro-olfactogram (EOG), and olfactory tissue was processed for histology and immunohistochemical staining. RESULTS: TNF-α was dramatically induced in IOI-TNFR1 knockout mice, but the olfactory epithelium did not show inflammation. EOG responses were normal after either 2 or 8 weeks of TNF-α expression. Ovalbumin-sensitized TNFR1 knockout mice developed markedly diminished eosinophilic inflammatory infiltration. CONCLUSION: Genetic deletion of TNFR1 completely blocks TNF-α-induced inflammation and reduces allergen-induced inflammation. Preserved EOG responses suggest a TNFR1-dependent mechanism of TNF-α-induced olfactory neuron dysfunction.

Innate myeloid cell TNFR1 mediates first line defence against primary Mycobacterium tuberculosis infection.

TNF is crucial for controlling Mycobacterium tuberculosis infection and understanding how will help immunomodulating the host response. Here we assessed the contribution of TNFR1 pathway from innate myeloid versus T cells. We first established the prominent role of TNFR1 in haematopoietic cells for controlling M. tuberculosis in TNFR1 KO chimera mice. Further, absence of TNFR1 specifically on myeloid cells (M-TNFR1 KO) recapitulated the uncontrolled M. tuberculosis infection seen in fully TNFR1 deficient mice, with increased bacterial burden, exacerbated lung inflammation, and rapid death. Pulmonary IL-12p40 over-expression was attributed to a prominent CD11b(+) Gr1(high) cell population in infected M-TNFR1 KO mice. By contrast, absence of TNFR1 on T-cells did not compromise the control of M. tuberculosis infection over 6-months. Thus, the protective TNF/TNFR1 pathway essential for controlling primary M. tuberculosis infection depends on innate macrophage and neutrophil myeloid cells, while TNFR1 pathway in T cells is dispensable.

Role of the TNF-α receptor type 1 on prostate carcinogenesis in knockout mice.

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.