The role of TNFR2+ Tregs in COVID-19: An overview and a potential therapeutic strategy.

COVID-19 is a multi-faceted disease ranging from asymptomatic to severely ill condition that primarily affects the lungs and could advance to other organs as well. It's causing factor, SARS-CoV-2 is recognized to develop robust cell-mediated immunity that responsible to either control or exaggerate the infection. As an important cell subset that control immune responses and are significantly dysregulated in COVID-19, Tregs is proposed to be considered for COVID-19 management. Among its hallmark, TNFR2 is recently recognized to play important role in the function and survival of Tregs. This review gathers available TNFR2 agonists to directly target Tregs as a potential approach to overcome immune dysregulation that affect the severity in COVID-19. Furthermore, this review performs a rigid body docking of TNF-TNFR2 interaction and such interaction with TNFR2 agonist to predict the optimal targeting approach.

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.

Transmembrane tumor necrosis factor alpha attenuates pressure-overload cardiac hypertrophy via tumor necrosis factor receptor 2.

Tumor necrosis factor-alpha (TNF-α) plays an important pathogenic role in cardiac hypertrophy and heart failure (HF); however, anti-TNF is paradoxically negative in clinical trials and even worsens HF, indicating a possible protective role of TNF-α in HF. TNF-α exists in transmembrane (tmTNF-α) and soluble (sTNF-α) forms. Herein, we found that TNF receptor 1 (TNFR1) knockout (KO) or knockdown (KD) by short hairpin RNA or small interfering RNA (siRNA) significantly alleviated cardiac hypertrophy, heart dysfunction, fibrosis, and inflammation with increased tmTNF-α expression, whereas TNFR2 KO or KD exacerbated the pathological phenomena with increased sTNF-α secretion in transverse aortic constriction (TAC)- and isoproterenol (ISO)-induced cardiac hypertrophy in vivo and in vitro, respectively, indicating the beneficial effects of TNFR2 associated with tmTNF-α. Suppressing TNF-α converting enzyme by TNF-α Protease Inhibitor-1 (TAPI-1) to increase endogenous tmTNF-α expression significantly alleviated TAC-induced cardiac hypertrophy. Importantly, direct addition of exogenous tmTNF-α into cardiomyocytes in vitro significantly reduced ISO-induced cardiac hypertrophy and transcription of the pro-inflammatory cytokines and induced proliferation. The beneficial effects of tmTNF-α were completely blocked by TNFR2 KD in H9C2 cells and TNFR2 KO in primary myocardial cells. Furthermore, we demonstrated that tmTNF-α displayed antihypertrophic and anti-inflammatory effects by activating the AKT pathway and inhibiting the nuclear factor (NF)-κB pathway via TNFR2. Our data suggest that tmTNF-α exerts cardioprotective effects via TNFR2. Specific targeting of tmTNF-α processing, rather than anti-TNF therapy, may be more useful for the treatment of hypertrophy and HF.

The Role of TNFR2 and DR3 in the In Vivo Expansion of Tregs in T Cell Depleting Transplantation Regimens.

Regulatory T cells (Tregs) are essential for the maintenance of tolerance to self and non-self through cell-intrinsic and cell-extrinsic mechanisms. Peripheral Tregs survival and clonal expansion largely depend on IL-2 and access to co-stimulatory signals such as CD28. Engagement of tumor necrosis factor receptor (TNFR) superfamily members, in particular TNFR2 and DR3, contribute to promote peripheral Tregs expansion and sustain their survival. This property can be leveraged to enhance tolerance to allogeneic transplants by tipping the balance of Tregs over conventional T cells during the course of immune reconstitution. This is of particular interest in peri-transplant tolerance induction protocols in which T cell depletion is applied to reduce the frequency of alloreactive T cells or in conditioning regimens that allow allogeneic bone marrow transplantation. These conditioning regimens are being implemented to limit long-term side effects of continuous immunosuppression and facilitate the establishment of a state of donor-specific tolerance. Lymphopenia-induced homeostatic proliferation in response to cytoreductive conditioning is a window of opportunity to enhance preferential expansion of Tregs during homeostatic proliferation that can be potentiated by agonist stimulation of TNFR.

Continuous infusion of an agonist of the tumor necrosis factor receptor 2 in the spinal cord improves recovery after traumatic contusive injury.

AIM: The activation of the TNFR2 receptor is beneficial in several pathologies of the central nervous system, and this study examines whether it can ameliorate the recovery process following spinal cord injury. METHODS: EHD2-sc-mTNFR2 , an agonist specific for TNFR2, was used to treat neurons exposed to high levels of glutamate in vitro. In vivo, it was infused directly to the spinal cord via osmotic pumps immediately after a contusion to the cord at the T9 level. Locomotion behavior was assessed for 6 weeks, and the tissue was analyzed (lesion size, RNA and protein expression, cell death) after injury. Somatosensory evoked potentials were also measured in response to hindlimb stimulation. RESULTS: The activation of TNFR2 protected neurons from glutamate-mediated excitotoxicity through the activation of phosphoinositide-3 kinase gamma in vitro and improved the locomotion of animals following spinal cord injury. The extent of the injury was not affected by infusing EHD2-sc-mTNFR2 , but higher levels of neurofilament H and 2', 3'-cyclic-nucleotide 3'-phosphodiesterase were observed 6 weeks after the injury. Finally, the activation of TNFR2 after injury increased the neural response recorded in the cortex following hindlimb stimulation. CONCLUSION: The activation of TNFR2 in the spinal cord following contusive injury leads to enhanced locomotion and better cortical responses to hindlimb stimulation.

TNF-TNFR2 Signaling Inhibits Th2 and Th17 Polarization and Alleviates Allergic Airway Inflammation.

BACKGROUND: TNF-TNFR2 signaling has been indicated to be involved in CD4+ T lymphocyte differentiation. However, its role in allergic airway inflammation is not well understood. OBJECTIVES: The aim of this study was to investigate the role of TNF-TNFR2 signaling in allergic airway inflammation. METHODS AND RESULTS: In this study, we used an allergen-induced asthma model to show that TNF-TNFR2 signaling alleviated allergic airway inflammation by reducing the airway infiltration of eosinophils and neutrophils. Activated TNF-TNFR2 signaling decreased the expression of Th2 and Th17 cytokines in serum and bronchoalveolar lavage fluid. Furthermore, TNF-TNFR2 signaling inhibited Th2 and Th17 polarization but promoted Th1 and CD4+CD25+ T cell differentiation in vivo. CONCLUSIONS: Our study indicates that TNF-TNFR2 signaling alleviates allergic airway inflammation through inhibition of Th2 and Th17 cell differentiation.

Intrinsic TNFR2 signaling in T regulatory cells provides protection in CNS autoimmunity.

TNF is a multifunctional cytokine involved in autoimmune disease pathogenesis that exerts its effects through two distinct TNF receptors, TNFR1 and TNFR2. While TNF- and TNFR1-deficient (but not TNFR2-deficient) mice show very similar phenotypes, the significance of TNFR2 signaling in health and disease remains incompletely understood. Recent studies implicated the importance of the TNF/TNFR2 axis in T regulatory (Treg) cell functions. To definitively ascertain the significance of TNFR2 signaling, we generated and validated doubly humanized TNF/TNFR2 mice, with the option of conditional inactivation of TNFR2. These mice carry a functional human TNF-TNFR2 (hTNF-hTNFR2) signaling module and provide a useful tool for comparative evaluation of TNF-directed biologics. Conditional inactivation of TNFR2 in FoxP3+ cells in doubly humanized TNF/TNFR2 mice down-regulated the expression of Treg signature molecules (such as FoxP3, CD25, CTLA-4, and GITR) and diminished Treg suppressive function in vitro. Consequently, Treg-restricted TNFR2 deficiency led to significant exacerbation of experimental autoimmune encephalomyelitis (EAE), accompanied by reduced capacity to control Th17-mediated immune responses. Our findings expose the intrinsic and beneficial effects of TNFR2 signaling in Treg cells that could translate into protective functions in vivo, including treatment of autoimmunity.

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.

Exogenous TNFR2 activation protects from acute GvHD via host T reg cell expansion.

Donor CD4(+)Foxp3(+) regulatory T cells (T reg cells) suppress graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (HCT [allo-HCT]). Current clinical study protocols rely on the ex vivo expansion of donor T reg cells and their infusion in high numbers. In this study, we present a novel strategy for inhibiting GvHD that is based on the in vivo expansion of recipient T reg cells before allo-HCT, exploiting the crucial role of tumor necrosis factor receptor 2 (TNFR2) in T reg cell biology. Expanding radiation-resistant host T reg cells in recipient mice using a mouse TNFR2-selective agonist before allo-HCT significantly prolonged survival and reduced GvHD severity in a TNFR2- and T reg cell-dependent manner. The beneficial effects of transplanted T cells against leukemia cells and infectious pathogens remained unaffected. A corresponding human TNFR2-specific agonist expanded human T reg cells in vitro. These observations indicate the potential of our strategy to protect allo-HCT patients from acute GvHD by expanding T reg cells via selective TNFR2 activation in vivo.

Expression of TNFR2 by regulatory T cells in peripheral blood is correlated with clinical pathology of lung cancer patients.

CD4(+)FoxP3(+) regulatory T cells (Tregs) represent a major cellular mediator of cancer immune evasion. The expression of tumor necrosis factor receptor type II (TNFR2) on Tregs is reported to identify the maximally suppressive Treg population in both mice and human. We therefore investigated the phenotype and function of TNFR2(+) Tregs present in the peripheral blood (PB) of 43 lung cancer patients. Further, the association of TNFR2 expression on Tregs with clinicopathological factors was analyzed. The results showed that in the PB of lung cancer patients, Tregs expressed markedly higher levels of TNFR2 than conventional T cells (Tconvs). Expression of TNFR2 appeared to correlate better than CD25(+) and CD127(-) with FoxP3 expression. PB TNFR2(+) Tregs in lung cancer patients were more proliferative and expressed higher levels of the immunosuppressive molecule CTLA-4, and consequently more potently suppressed IFNγ production by cocultured CD8 CTLs. More importantly, higher TNFR2 expression levels on Tregs were associated with lymphatic invasion, distant metastasis and more advanced clinical stage of lung cancer patients. Therefore, our study suggests that TNFR2(+) Tregs play a role in promoting tumor progressive metastasis and expression of TNFR2 by PB Tregs may prove to be a useful prognostic marker in lung cancer patients.

Tumor necrosis factor reduces the amplitude of rat cortical spreading depression in vivo.

OBJECTIVE: Brain damage and ischemia often trigger cortical spreading depression (CSD), which aggravates brain damage. The proinflammatory cytokine tumor necrosis factor (TNF) is significantly upregulated during brain damage, but it is unknown whether TNF influences spreading depression in cerebral cortex in vivo. This question is important because TNF not only furthers inflammatory reactions but might also be neuroprotective. Here we tested the hypothesis that TNF affects CSD, and we explored the direction in which CSD is modified by TNF. METHODS: CSD, elicited by pressure microinjection of KCl, was recorded in anesthetized rats and mice. TNF was administered locally into a trough, providing local TNF treatment of a cortical area. For further analysis, antibodies to TNF receptor (TNFR) 1 or 2 were applied, or CSD was monitored in TNFR1 and TNFR2 knockout mice. γ-Aminobutyric acid (GABA)A receptors were blocked by bicuculline. Immunohistochemistry localized the cortical expression of TNFR1 and TNFR2. RESULTS: Local application of TNF to the cortex reduced dose-dependently the amplitude of CSD. This effect was prevented by blockade or knockout of TNFR2 but not by blockade or knockout of TNFR1. TNFR2 was localized at cortical neurons including parvalbumin-positive inhibitory interneurons, and blockade of GABAA receptors by bicuculline prevented the reduction of CSD amplitudes by TNF. INTERPRETATION: We identified a functional link between TNF and CSD. TNF activates TNFR2 in cortical inhibitory interneurons. The resulting release of GABA reduces CSD amplitudes. In this manner, TNF might be neuroprotective in pathological conditions.

Distinct role of tumor necrosis factor receptor subtypes 1 and 2 in the red nucleus in the development of neuropathic pain.

Previous studies have demonstrated that tumor necrosis factor-alpha (TNF-α) in the red nucleus (RN) plays a facilitated role in the development of neuropathic pain. Here, the protein levels and roles of two different TNF receptors, p55 type 1 (TNFR1) and p75 type 2 (TNFR2), in the RN of rats with spared nerve injury (SNI) were investigated. Immunohistochemistry demonstrated that both TNFR1 and TNFR2 were significantly increased in the RN of rats with SNI compared with sham-operated and normal rats. The up-regulation of TNFR1 occurred at two weeks after SNI, while TNFR2 had markedly increased already at one week and began to decrease at two weeks after SNI. Microinjection of different doses (500, 250 and 100ng) of anti-TNFR1 antibody (anti-TNFR1-Ab) or anti-TNFR2-Ab into the RN contralateral to the nerve injury side dose-dependently increased the paw withdrawal threshold of rats, as assessed using von Frey filaments. The analgesic effects produced by anti-TNFR1-Ab at one week and two weeks after SNI did not show significant difference. However, the analgesic effect produced by anti-TNFR2-Ab at two weeks after SNI was significantly lower and shorter than that produced at one week after SNI. Combined injection of anti-TNFR1-Ab and anti-TNFR2-Ab (500ng for each antibody) into the RN generated a relatively faster and longer analgesic effect compared with single using of anti-TNFR1-Ab or anti-TNFR2-Ab. These results support that TNF-α in the RN plays a crucial role in regulating neuropathic pain, and suggest that the algesic effect of TNF-α is transmitted through both TNFR1 and TNFR2. TNFR1 has equally important role in the early development and later maintenance of neuropathic pain, while TNFR2 is more inclined to play a role in the early development of neuropathic pain.

Transmembrane TNF-α promotes suppressive activities of myeloid-derived suppressor cells via TNFR2.

It has been reported that TNFR2 is involved in regulatory T cell induction and myeloid-derived suppressor cell (MDSC) accumulation, two kinds of immunosuppressive cells contributing to tumor immune evasion. Because transmembrane TNF-α (tmTNF-α) is the primary ligand for TNFR2, we hypothesized that tmTNF-α is mainly responsible for the activation of MDSCs. Indeed, we found that tmTNF-α, rather than secretory TNF-α (sTNF-α), activated MDSCs with enhanced suppressive activities, including upregulating arginase-1 and inducible NO synthase transcription, promoting secretion of NO, reactive oxygen species, IL-10, and TGF-ß, and enhancing inhibition of lymphocyte proliferation. This effect of tmTNF-α was mediated by TNFR2, as TNFR2 deficiency significantly impaired tmTNF-α-induced release of IL-10 and NO and inhibition of T cell proliferation by MDSC supernatant. Furthermore, tmTNF-α caused p38 phosphorylation and NF-κB activation, whereas inhibition of NF-κB or p38 with an inhibitor pyrrolidine dithiocarbamate or SB203580 abrogated tmTNF-α-mediated increased suppression of lymphocyte proliferation by MDSCs. Consistently, our in vivo study showed that ectopic expression of uncleavable tmTNF-α mutant by 4T1 cells significantly promoted tumor progression and angiogenesis, accompanied with more accumulation of MDSCs and regulatory T cells in the tumor site, increased production of NO, IL-10, and TGF-ß, as well as poor lymphocyte infiltration. In contrast, enforced expression of sTNF-α mutant by 4T1 cells that only released sTNF-α without expression of surface tmTNF-α markedly reduced MDSC accumulation and induced more lymphocyte infiltration instead, showing obvious tumor regression. Our data suggest that tmTNF-α acts as a potent activator of MDSCs via TNFR2 and reveals another novel immunosuppressive effect of this membrane molecule that promotes tumor immune escape.

Therapeutic effects of p75 tumor necrosis factor receptor monoclonal antibody on a rat model of traumatic arthritis.

BACKGROUND: The aim of the present study was to investigate the therapeutic effects of p75 tumor necrosis factor receptor monoclonal antibody D8F2 on a traumatic arthritis model in rats, and to explore the underlying mechanism. METHODS: Forty male Sprague Dawley rats were randomly divided into five groups: (A) sham operation control group, (B) traumatic arthritis model group, (C) low-dose D8F2 group (1 mg/kg), (D) medium-dose D8F2 group (3 mg/kg), and (E) high-dose D8F2 group (10 mg/kg). Joint fluid samples were collected at 72 h after surgery, and enzyme-linked immunosorbent assay was performed to measure the following inflammatory factors: tumor necrosis factor α (TNF-α) and interleukin 1ß. One week after the surgery, rats were killed, and immunohistochemical staining was applied to detect the matrix metalloproteinase (MMP1 and MMP3) expression in the synovium. In cultured synovial fibroblast experiments, the D8F2-induced ubiquitination of TNF receptor-associated factor 2 (TRAF2) was examined by immunoprecipitation, and nuclear translocation of p65 nuclear factor-κB (p65NF-κB) mediated by TNF-α and D8F2 was analyzed by western blotting. RESULTS: In the traumatic arthritis model group, the inflammatory factors and MMPs were significantly increased relative to the sham operation control group (P < 0.05), whereas D8F2 could downregulate these factors in a dose-dependent manner (P < 0.05). The results from in vitro studies indicated that D8F2 can induce TRAF2 ubiquitination and inhibit the nuclear translocation of p65NF-κB mediated by TNF-α. CONCLUSIONS: p75 Tumor necrosis factor receptor monoclonal antibody has a therapeutic effect on traumatic arthritis, which may occur via the downregulation of inflammatory factors and MMPs at the transcription level because of TRAF2 degradation and inhibited activation of NF-κB.

TNF receptor 2 protects oligodendrocyte progenitor cells against oxidative stress.

The neuroprotective role of TNF receptor 2 (TNFR2) has been shown in various studies. However, a direct role of TNFR2 in oligodendrocyte function has not yet been demonstrated. Using primary oligodendrocytes of transgenic mice expressing human TNFR2, we show here that TNFR2 is primarily expressed on oligodendrocyte progenitor cells. Interestingly, preconditioning with a TNFR2 agonist protects these cells from oxidative stress, presumably by increasing the gene expression of distinct anti-apoptotic and detoxifying proteins, thereby providing a potential mechanism for the neuroprotective role of TNFR2 in oligodendrocyte progenitor cells.

Distinct contributions of TNF receptor 1 and 2 to TNF-induced glomerular inflammation in mice.

TNF is an important mediator of glomerulonephritis. The two TNF-receptors TNFR1 and TNFR2 contribute differently to glomerular inflammation in vivo, but specific mechanisms of TNFR-mediated inflammatory responses in glomeruli are unknown. We investigated their expression and function in murine kidneys, isolated glomeruli ex vivo, and glomerular cells in vitro. In normal kidney TNFR1 and TNFR2 were preferentially expressed in glomeruli. Expression of both TNFRs and TNF-induced upregulation of TNFR2 mRNA was confirmed in murine glomerular endothelial and mesangial cell lines. In vivo, TNF exposure rapidly induced glomerular accumulation of leukocytes. To examine TNFR-specific inflammatory responses in intrinsic glomerular cells but not infiltrating leukocytes we performed microarray gene expression profiling on intact glomeruli isolated from wildtype and Tnfr-deficient mice following exposure to soluble TNF ex vivo. Most TNF-induced effects were exclusively mediated by TNFR1, including induced glomerular expression of adhesion molecules, chemokines, complement factors and pro-apoptotic molecules. However, TNFR2 contributed to TNFR1-dependent mRNA expression of inflammatory mediators in glomeruli when exposed to low TNF concentrations. Chemokine secretion was absent in TNF-stimulated Tnfr1-deficient glomeruli, but also significantly decreased in glomeruli lacking TNFR2. In vivo, TNF-induced glomerular leukocyte infiltration was abrogated in Tnfr1-deficient mice, whereas Tnfr2-deficiency decreased mononuclear phagocytes infiltrates, but not neutrophils. These data demonstrate that activation of intrinsic glomerular cells by soluble TNF requires TNFR1, whereas TNFR2 is not essential, but augments TNFR1-dependent effects. Previously described TNFR2-dependent glomerular inflammation may therefore require TNFR2 activation by membrane-bound, but not soluble TNF.

Tumour necrosis factor--induced loss of intestinal barrier function requires TNFR1 and TNFR2 signalling in a mouse model of total parenteral nutrition.

Tumour necrosis factor-α (TNF-α) has been reported to play a central role in intestinal barrier dysfunction in many diseases; however, the precise role of the TNF-α receptors (TNFRs) has not been well defined using in vivo models. Our previous data showed that enteral nutrient deprivation or total parenteral nutrition (TPN) led to a loss of intestinal epithelial barrier function (EBF), with an associated upregulation of TNF-α and TNFR1. In this study, we hypothesized that TNF-α plays an important role in TPN-associated EBF dysfunction. Using a mouse TPN model, we explored the relative roles of TNFR1 vs. TNFR2 in mediating this barrier loss. C57/BL6 mice underwent intravenous cannulation and were given enteral nutrition or TPN for 7 days. Tumour necrosis factor-α receptor knockout (KO) mice, including TNFR1KO, TNFR2KO or TNFR1R2 double KO (DKO), were used. Outcomes included small intestine transepithelial resistance (TER) and tracer permeability, junctional protein zonula occludens-1, occludin, claudins and E-cadherin expression. In order to address the dependence of EBF on TNF-α further, exogenous TNF-α and pharmacological blockade of TNF-α (Etanercept) were also performed. Total parenteral nutrition led to a loss of EBF, and this was almost completely prevented in TNFR1R2DKO mice and partly prevented in TNFR1KO mice but not in TNFR2KO mice. The TPN-associated downregulation of junctional protein expression and junctional assembly was almost completely prevented in the TNFR1R2DKO group. Blockade of TNF-α also prevented dysfunction of the EBF and junctional protein losses in mice undergoing TPN. Administration of TPN upregulated the downstream nuclear factor-B and myosin light-chain kinase (MLCK) signalling, and these changes were almost completely prevented in TNFR1R2DKO mice, as well as with TNF-α blockade, but not in TNFR1KO or TNFR2KO TPN groups. Tumour necrosis factor-α is a critical factor for TPN-associated epithelial barrier dysfunction, and both TNFR1 and TNFR2 are involved in EBF loss. Nuclear factor-B and MLCK signalling appear to be important downstream mediators involved in this TNF-α signalling process.