TNF receptor 2 knockout mouse had reduced lung cancer growth and schizophrenia-like behavior through a decrease in TrkB-dependent BDNF level.

The relationship between schizophrenia (SCZ) and cancer development remains controversial. Based on the disease-gene association platform, it has been revealed that tumor necrosis factor receptor (TNFR) could be an important mediatory factor in both cancer and SCZ development. TNF-α also increases the expression of brain-derived neurotrophic factor (BDNF) and tropomyosin receptor kinase B (TrkB) in the development of SCZ and tumor, but the role of TNFR in mediating the association between the two diseases remains unclear. We studied the vital roles of TNFR2 in the progression of tumor and SCZ-like behavior using A549 lung cancer cell xenografted TNFR2 knockout mice. TNFR2 knockout mice showed significantly decreased tumor size and weight as well as schizophrenia-like behaviors compared to wild-type mice. Consistent with the reduced tumor growth and SCZ-like behaviors, the levels of TrkB and BDNF expression were significantly decreased in the lung tumor tissues and pre-frontal cortex of TNFR2 knockout mice. However, intravenous injection of BDNF (160 µg/kg) to TNFR2 knockout mice for 4 weeks increased tumor growth and SCZ-like behaviors as well as TrkB expression. In in vitro study, significantly decreased cell growth and expression of TrkB and BDNF by siTNFR2 transfection were found in A549 lung cancer cells. However, the addition of BDNF (100 ng/ml) into TNFR2 siRNA transfected A549 lung cancer cells recovered cell growth and the expression of TrkB. These results suggest that TNFR2 could be an important factor in mediating the comorbidity between lung tumor growth and SCZ development through increased TrkB-dependent BDNF levels.

Transmembrane TNF-TNFR2 signaling as a critical immunoregulatory node in pancreatic cancer.

Pancreatic cancer is characterized by extreme therapeutic resistance. In pancreatic cancers harboring high-risk genomes, we describe that cancer cell-neutrophil signaling circuitry provokes neutrophil-derived transmembrane (tm)TNF-TNFR2 interactions that dictate inflammatory polarization in cancer-associated fibroblasts and T-cell dysfunction - two hallmarks of therapeutic resistance. Targeting tmTNF-TNFR2 signaling may sensitize pancreatic cancer to chemo±immunotherapy.

TNFRSF1B Gene Variants in Clinicopathological Aspects and Prognosis of Patients with Cutaneous Melanoma.

Regulatory T lymphocytes play a critical role in immune regulation and are involved in the aberrant cell elimination by facilitating tumor necrosis factor connection to the TNFR2 receptor, encoded by the TNFRSF1B polymorphic gene. We aimed to examine the effects of single nucleotide variants TNFRSF1B c.587T>G, c.*188A>G, c.*215C>T, and c.*922C>T on the clinicopathological characteristics and survival of cutaneous melanoma (CM) patients. Patients were genotyped using RT-PCR. TNFRSF1B levels were measured using qPCR. Luciferase reporter assay evaluated the interaction of miR-96 and miR-1271 with the 3'-UTR of TNFRSF1B. The c.587TT genotype was more common in patients younger than 54 years old than in older patients. Patients with c.*922CT or TT, c.587TG or GG + c.*922CT or TT genotypes, as well as those with the haplotype TATT, presented a higher risk of tumor progression and death due to the disease effects. Individuals with the c.*922TT genotype had a higher TNFRSF1B expression than those with the CC genotype. miR-1271 had less efficient binding with the 3'-UTR of the T allele when compared with the C allele of the SNV c.*922C>T. Our findings, for the first time, demonstrate that TNFRSF1B c.587T>G and c.*922C>T variants can serve as independent prognostic factors in CM patients.

An immunocytokine consisting of a TNFR2 agonist and TNFR2 scFv enhances the expansion of regulatory T cells through TNFR2 clustering.

Regulatory T cells (Tregs) are lymphocytes that play a central role in peripheral immune tolerance. Tregs are promising targets for the prevention and suppression of autoimmune diseases, allergies, and graft-versus-host disease, and treatments aimed at regulating their functions are being developed. In this study, we created a new modality consisting of a protein molecule that suppressed excessive immune responses by effectively and preferentially expanding Tregs. Recent studies reported that tumor necrosis factor receptor type 2 (TNFR2) expressed on Tregs is involved in the proliferation and activation of Tregs. Therefore, we created a functional immunocytokine, named TNFR2-ICK-Ig, consisting of a fusion protein of an anti-TNFR2 single-chain Fv (scFv) and a TNFR2 agonist TNF-α mutant protein, as a new modality that strongly enhances TNFR2 signaling. The formation of agonist-receptor multimerization (TNFR2 cluster) is effective for the induction of a strong TNFR2 signal, similar to the TNFR2 signaling mechanism exhibited by membrane-bound TNF. TNFR2-ICK-Ig improved the TNFR2 signaling activity and promoted TNFR2 cluster formation compared to a TNFR2 agonist TNF-α mutant protein that did not have an immunocytokine structure. Furthermore, the Treg expansion efficiency was enhanced. TNFR2-ICK-Ig promotes its effects via scFv, which crosslinks receptors whereas the agonists transmit stimulatory signals. Therefore, this novel molecule expands Tregs via strong TNFR2 signaling by the formation of TNFR2 clustering.

Microglial TNFR2 signaling regulates the inflammatory response after CNS injury in a sex-specific fashion.

Microglia, the resident immune cells of the central nervous system (CNS), play a major role in damage progression and tissue remodeling after acute CNS injury, including ischemic stroke (IS) and spinal cord injury (SCI). Understanding the molecular mechanisms regulating microglial responses to injury may thus reveal novel therapeutic targets to promote CNS repair. Here, we investigated the role of microglial tumor necrosis factor receptor 2 (TNFR2), a transmembrane receptor previously associated with pro-survival and neuroprotective responses, in shaping the neuroinflammatory environment after CNS injury. By inducing experimental IS and SCI in Cx3cr1CreER:Tnfrsf1bfl/fl mice, selectively lacking TNFR2 in microglia, and corresponding Tnfrsf1bfl/fl littermate controls, we found that ablation of microglial TNFR2 significantly reduces lesion size and pro-inflammatory cytokine levels, and favors infiltration of leukocytes after injury. Interestingly, these effects were paralleled by opposite sex-specific modifications of microglial reactivity, which was found to be limited in female TNFR2-ablated mice compared to controls, whereas it was enhanced in males. In addition, we show that TNFR2 protein levels in the cerebrospinal fluid (CSF) of human subjects affected by IS and SCI, as well as healthy donors, significantly correlate with disease stage and severity, representing a valuable tool to monitor the inflammatory response after acute CNS injury. Hence, these results advance our understanding of the mechanisms regulating microglia reactivity after acute CNS injury, aiding the development of sex- and microglia-specific, personalized neuroregenerative strategies.

Deletion of the non-adjacent genes UL148 and UL148D impairs human cytomegalovirus-mediated TNF receptor 2 surface upregulation.

Human cytomegalovirus (HCMV) is a prototypical ß-herpesvirus which frequently causes morbidity and mortality in individuals with immature, suppressed, or senescent immunity. HCMV is sensed by various pattern recognition receptors, leading to the secretion of pro-inflammatory cytokines including tumor necrosis factor alpha (TNFα). TNFα binds to two distinct trimeric receptors: TNF receptor (TNFR) 1 and TNFR2, which differ in regard to their expression profiles, affinities for soluble and membrane-bound TNFα, and down-stream signaling pathways. While both TNF receptors engage NFκB signaling, only the nearly ubiquitously expressed TNFR1 exhibits a death domain that mediates TRADD/FADD-dependent caspase activation. Under steady-state conditions, TNFR2 expression is mainly restricted to immune cells where it predominantly submits pro-survival, proliferation-stimulating, and immune-regulatory signals. Based on the observation that HCMV-infected cells show enhanced binding of TNFα, we explored the interplay between HCMV and TNFR2. As expected, uninfected fibroblasts did not show detectable levels of TNFR2 on the surface. Intriguingly, however, HCMV infection increased TNFR2 surface levels of fibroblasts. Using HCMV variants and BACmid-derived clones either harboring or lacking the ULb' region, an association between TNFR2 upregulation and the presence of the ULb' genome region became evident. Applying a comprehensive set of ULb' gene block and single gene deletion mutants, we observed that HCMV mutants in which the non-adjacent genes UL148 or UL148D had been deleted show an impaired ability to upregulate TNFR2, coinciding with an inverse regulation of TACE/ADAM17.

Bivalent structure of a TNFR2-selective and agonistic TNF-α mutein Fc-fusion protein enhances the expansion activity of regulatory T cells.

Recently, TNF receptor type 2 (TNFR2) signaling was found to be involved in the proliferation and activation of regulatory T cells (Tregs), a subpopulation of lymphocytes that suppress immune responses. Tregs mediate peripheral immune tolerance, and the disruption of their functions causes autoimmune diseases or allergy. Therefore, cell expanders or regulators of Tregs that control immunosuppressive activity can be used to treat these diseases. We focused on TNFR2, which is preferentially expressed on Tregs, and created tumor necrosis factor-α (TNF-α) muteins that selectively activate TNFR2 signaling in mice and humans, termed R2agoTNF and R2-7, respectively. In this study, we attempted to optimize the structure of muteins to enhance their TNFR2 agonistic activity and stability in vivo by IgG-Fc fusion following single-chain homo-trimerization. The fusion protein, scR2agoTNF-Fc, enhanced the expansion of CD4+CD25+ Tregs and CD4+Foxp3+ Tregs and contributed to their immunosuppressive activity ex vivo and in vivo in mice. The prophylactic administration of scR2agoTNF-Fc suppressed inflammation in contact hypersensitivity and arthritis mouse models. Furthermore, scR2-7-Fc preferentially expanded Tregs in human peripheral blood mononuclear cells via TNFR2. These TNFR2 agonist-Fc fusion proteins, which have bivalent structures, are novel Treg expanders.

Activation of TNF Receptor 2 Improves Synaptic Plasticity and Enhances Amyloid-ß Clearance in an Alzheimer's Disease Mouse Model with Humanized TNF Receptor 2.

BACKGROUND: Tumor necrosis factor-alpha (TNF-α) is a master cytokine involved in a variety of inflammatory and neurological diseases, including Alzheimer's disease (AD). Therapies that block TNF-α proved ineffective as therapeutic for neurodegenerative diseases, which might be explained by the opposing functions of the two receptors of TNF (TNFRs): while TNFR1 stimulation mediates inflammatory and apoptotic pathways, activation of TNFR2 is related to neuroprotection. Despite the success of targeting TNFR2 in a transgenic AD mouse model, research that better mimics the human context is lacking. OBJECTIVE: The aim of this study is to investigate whether stimulation of TNFR2 with a TNFR2 agonist is effective in activating human TNFR2 and attenuating AD neuropathology in the J20xhuTNFR2-k/i mouse model. METHODS: Transgenic amyloid-ß (Aß)-overexpressing mice containing a human extracellular TNFR2 domain (J20xhuTNFR2-k/i) were treated with a TNFR2 agonist (NewStar2). After treatment, different behavioral tests and immunohistochemical analysis were performed to assess different parameters, such as cognitive functions, plaque deposition, synaptic plasticity, or microglial phagocytosis. RESULTS: Treatment with NewStar2 in J20xhuTNFR2-k/i mice resulted in a drastic decrease in plaque load and beta-secretase 1 (BACE-1) compared to controls. Moreover, TNFR2 stimulation increased microglial phagocytic activity, leading to enhanced Aß clearance. Finally, activation of TNFR2 rescued cognitive impairments and improved synaptic plasticity. CONCLUSION: Our findings demonstrate that activation of human TNFR2 ameliorates neuropathology and improves cognitive functions in an AD mouse model. Moreover, our study confirms that the J20xhuTNFR2-k/i mouse model is suitable for testing human TNFR2-specific compounds.

Distinct immune modulatory roles of regulatory T cells in gut versus joint inflammation in TNF-driven spondyloarthritis.

OBJECTIVES: Gut and joint inflammation commonly co-occur in spondyloarthritis (SpA) which strongly restricts therapeutic modalities. The immunobiology underlying differences between gut and joint immune regulation, however, is poorly understood. We therefore assessed the immunoregulatory role of CD4+FOXP3+ regulatory T (Treg) cells in a model of Crohn's-like ileitis and concomitant arthritis. METHODS: RNA-sequencing and flow cytometry was performed on inflamed gut and joint samples and tissue-derived Tregs from tumour necrosis factor (TNF)∆ARE mice. In situ hybridisation of TNF and its receptors (TNFR) was applied to human SpA gut biopsies. Soluble TNFR (sTNFR) levels were measured in serum of mice and patients with SpA and controls. Treg function was explored by in vitro cocultures and in vivo by conditional Treg depletion. RESULTS: Chronic TNF exposure induced several TNF superfamily (TNFSF) members (4-1BBL, TWEAK and TRAIL) in synovium and ileum in a site-specific manner. Elevated TNFR2 messenger RNA levels were noted in TNF∆ARE/+ mice leading to increased sTNFR2 release. Likewise, sTNFR2 levels were higher in patients with SpA with gut inflammation and distinct from inflammatory and healthy controls. Tregs accumulated at both gut and joints of TNF∆ARE mice, yet their TNFR2 expression and suppressive function was significantly lower in synovium versus ileum. In line herewith, synovial and intestinal Tregs displayed a distinct transcriptional profile with tissue-restricted TNFSF receptor and p38MAPK gene expression. CONCLUSIONS: These data point to profound differences in immune-regulation between Crohn's ileitis and peripheral arthritis. Whereas Tregs control ileitis they fail to dampen joint inflammation. Synovial resident Tregs are particularly maladapted to chronic TNF exposure.

TNFα Causes a Shift in Gene Expression of TNFRSF1A and TNFRSF1B Isoforms.

Alternative splicing is a part of mRNA processing that expands the diversity of proteins encoded by a single gene. Studying the full range of proteins-products of translation of alternatively spliced mRNA is extremely important for understanding the interactions between receptor proteins and ligands since different receptor protein isoforms can provide variation in the activation of signaling pathways. In this study, we investigated the expression of isoforms of TNFR1 and TNFR2 receptors before and after exposure to TNFα in two cell lines that had previously demonstrated diverse effects on cell proliferation under TNFα incubation using RT-qPCR. We found that after incubation with TNFα: (1) expression of isoform 3 of the TNFRSF1A gene was increased in both cell lines; (2) the cell line with increased proliferation, K562, had decreased expression of isoforms 1 and 4 of the TNFRSF1A gene and expression of isoform 2 of TNFRSF1B gene was absent at all; (3) the cell line with decreased proliferation-MCF-7 had significantly increased expression of isoform 2 of TNFRSF1B gene. Thus, we can conclude that TNFα exposure to the K562 and MCF-7 cell lines leads to changes in the expression of TNFα receptor isoforms, which, in turn, can appear via diverse proliferative effects.

Co-modulation of TNFR1 and TNFR2 in an animal model of multiple sclerosis.

BACKGROUND: Tumour necrosis factor (TNF) is a pleiotropic cytokine and master regulator of the immune system. It acts through two receptors resulting in often opposing biological effects, which may explain the lack of therapeutic potential obtained so far in multiple sclerosis (MS) with non-receptor-specific anti-TNF therapeutics. Under neuroinflammatory conditions, such as MS, TNF receptor-1 (TNFR1) is believed to mediate the pro-inflammatory activities associated with TNF, whereas TNF receptor-2 (TNFR2) may instead induce anti-inflammatory effects as well as promote remyelination and neuroprotection. In this study, we have investigated the therapeutic potential of blocking TNFR1 whilst simultaneously stimulating TNFR2 in a mouse model of MS. METHODS: Experimental autoimmune encephalomyelitis (EAE) was induced with myelin oligodendrocyte glycoprotein (MOG35-55) in humanized TNFR1 knock-in mice. These were treated with a human-specific TNFR1-selective antagonistic antibody (H398) and a mouse-specific TNFR2 agonist (EHD2-sc-mTNFR2), both in combination and individually. Histopathological analysis of spinal cords was performed to investigate demyelination and inflammatory infiltration, as well as axonal and neuronal degeneration. Retinas were examined for any protective effects on retinal ganglion cell (RGC) degeneration and neuroprotective signalling pathways analysed by Western blotting. RESULTS: TNFR modulation successfully ameliorated symptoms of EAE and reduced demyelination, inflammatory infiltration and axonal degeneration. Furthermore, the combinatorial approach of blocking TNFR1 and stimulating TNFR2 signalling increased RGC survival and promoted the phosphorylation of Akt and NF-κB, both known to mediate neuroprotection. CONCLUSION: These results further support the potential of regulating the balance of TNFR signalling, through the co-modulation of TNFR1 and TNFR2 activity, as a novel therapeutic approach in treating inflammatory demyelinating disease.

TNFR2 deficiency impairs the growth of mouse colon cancer.

Objective: Tumor necrosis factor (TNF) receptor type II (TNFR2) is expressed by a wide spectrum of tumor cells including colon cancer, non-Hodgkin lymphoma, myeloma, renal carcinoma and ovarian cancer, and its exact role remains to be fully understood. In this study, we examined the effect of genetic ablation of TNFR2 on in vitro and in vivo growth of mouse MC38 and CT26 colon cancer cells. Methods: CRISPR/Cas9 technology was used to knockout TNFR2 on mouse MC38 and CT26 colon cancer cells. In vitro growth and colony formation of wild-type (W.T.) and TNFR2 deficiency of MC38 and CT26 cells, as well as the potential mechanism, was studied. The growth of W.T. and TNFR2 deficient MC38 and CT26 tumors in mice and intratumoral CD8 CTLs were also examined. Results: TNFR2 deficiency impaired in vitro proliferation and colony formation of cancer cells. This was associated with the inhibition of protein kinase B (AKT) phosphorylation and enhanced autophagy-induced cell death. Moreover, deficiency of TNFR2 also markedly impaired in vivo growth of MC38 or CT26 in the syngeneic C57BL/6 mice or BALB/c mice, respectively, accompanied by the decrease in soluble TNFR2 levels in the circulation and the increase in the number of tumor-infiltrating IFNγ+ CD8 cells. Conclusion: TNFR2 plays a role in the growth of mouse colon cancers. Our study provides further experimental evidence to support the development of TNFR2 antagonistic agents in the treatment of cancer.

TNFR2 expression predicts the responses to immune checkpoint inhibitor treatments.

Immune checkpoint inhibitors (ICIs) by targeting PD-1/PD-L1 or CTLA-4 have markedly improved the outcome of cancer patients. However, most solid tumor patients can't benefit from such therapy. Identification of novel biomarkers to predict the responses of ICIs is crucial to enhance their therapeutic efficacy. TNFR2 is highly expressed by the maximally immunosuppressive subset of CD4+Foxp3+ regulatory T cells (Tregs), especially those present in tumor microenvironment (TME). Since Tregs represent a major cellular mechanism in tumor immune evasion, TNFR2 may be a useful biomarker to predict the responses to ICIs therapy. This notion is supported by our analysis of the computational tumor immune dysfunction and exclusion (TIDE) framework from published single-cell RNA-seq data of pan-cancer databases. The results show that, as expected, TNFR2 is highly expressed by tumor-infiltrating Tregs. Interestingly, TNFR2 is also expressed by the exhausted CD8 T cells in breast cancer (BRCA), hepatocellular carcinoma (HCC), lung squamous cell carcinoma (LUSC), and melanoma (MELA). Importantly, high expression of TNFR2 is associated with poor responses to the treatment with ICIs in BRCA, HCC, LUSC, and MELA. In conclusion, the expression of TNFR2 in TME may be a reliable biomarker for the precision of ICIs treatment of cancer patients, and this idea merits further research.

Interaction between tumor cell TNFR2 and monocyte membrane-bound TNF-α triggers tumorigenic inflammation in neuroblastoma.

BACKGROUND: Tumor progression and resistance to therapy in children with neuroblastoma (NB), a common childhood cancer, are often associated with infiltration of monocytes and macrophages that produce inflammatory cytokines. However, the mechanism by which tumor-supportive inflammation is initiated and propagated remains unknown. Here, we describe a novel protumorigenic circuit between NB cells and monocytes that is triggered and sustained by tumor necrosis factor alpha (TNF-α). METHODS: We used NB knockouts (KOs) of TNF-α and TNFRSF1A mRNA (TNFR1)/TNFRSF1B mRNA (TNFR2) and TNF-α protease inbitor (TAPI), a drug that modulates TNF-α isoform expression, to assess the role of each component in monocyte-associated protumorigenic inflammation. Additionally, we employed NB-monocyte cocultures and treated these with clinical-grade etanercept, an Fc-TNFR2 fusion protein, to neutralize signaling by both membrane-bound (m) and soluble (s)TNF-α isoforms. Further, we treated NOD/SCID/IL2Rγ(null) mice carrying subcutaneous NB/human monocyte xenografts with etanercept and evaluated the impact on tumor growth and angiogenesis. Gene set enrichment analysis (GSEA) was used to determine whether TNF-α signaling correlates with clinical outcomes in patients with NB. RESULTS: We found that NB expression of TNFR2 and monocyte membrane-bound tumor necrosis factor alpha is required for monocyte activation and interleukin (IL)-6 production, while NB TNFR1 and monocyte soluble TNF-α are required for NB nuclear factor kappa B subunit 1 (NF-κB) activation. Treatment of NB-monocyte cocultures with clinical-grade etanercept completely abrogated release of IL-6, granulocyte colony-stimulating factor (G-CSF), IL-1α, and IL-1ß and eliminated monocyte-induced enhancement of NB cell proliferation in vitro. Furthermore, etanercept treatment inhibited tumor growth, ablated tumor angiogenesis, and suppressed oncogenic signaling in mice with subcutaneous NB/human monocyte xenografts. Finally, GSEA revealed significant enrichment for TNF-α signaling in patients with NB that relapsed. CONCLUSIONS: We have described a novel mechanism of tumor-promoting inflammation in NB that is strongly associated with patient outcome and could be targeted with therapy.

Expression of GnT-III decreases chemoresistance via negatively regulating P-glycoprotein expression: Involvement of the TNFR2-NF-κB signaling pathway.

The phenomenon of multidrug resistance (MDR) is called chemoresistance with respect to the treatment of cancer, and it continues to be a major challenge. The role of N-glycosylation in chemoresistance, however, remains poorly understood. Here, we established a traditional model for adriamycin resistance in K562 cells, which are also known as K562/adriamycin-resistant (ADR) cells. Lectin blot, mass spectrometry, and RT-PCR analysis showed that the expression levels of N-acetylglucosaminyltransferase III (GnT-III) mRNA and its products, bisected N-glycans, are significantly decreased in K562/ADR cells, compared with the levels in parent K562 cells. By contrast, the expression levels of both P-glycoprotein (P-gp) and its intracellular key regulator, NF-κB signaling, are significantly increased in K562/ADR cells. These upregulations were sufficiently suppressed by the overexpression of GnT-III in K562/ADR cells. We found that the expression of GnT-III consistently decreased chemoresistance for doxorubicin and dasatinib, as well as activation of the NF-κB pathway by tumor necrosis factor (TNF) α, which binds to two structurally distinct glycoproteins, TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2), on the cell surface. Interestingly, our immunoprecipitation analysis revealed that only TNFR2, but not TNFR1, contains bisected N-glycans. The lack of GnT-III strongly induced TNFR2's autotrimerization without ligand stimulation, which was rescued by the overexpression of GnT-III in K562/ADR cells. Furthermore, the deficiency of TNFR2 suppressed P-gp expression while it increased GnT-III expression. Taken together, these results clearly show that GnT-III negatively regulates chemoresistance via the suppression of P-gp expression, which is regulated by the TNFR2-NF/κB signaling pathway.

Tumor necrosis factor-alpha blockade suppresses BK polyomavirus replication.

PURPOSE: BK Polyomavirus (BKPyV) infection manifests as renal inflammation and can cause kidney damage. Tumor necrosis factor-α (TNF-α) is increased in renal inflammation and injury. The aim of this study was to investigate the effect of TNF-α blockade on BKPyV infection. METHODS: Urine specimens from 22 patients with BKPyV-associated nephropathy (BKPyVN) and 35 non-BKPyVN kidney transplant recipients were analyzed. RESULTS: We demonstrated increased urinary levels of TNF-α and its receptors, TNFR1 and TNFR2, in BKPyVN patients. Treating BKPyV-infected human proximal tubular cells (HRPTECs) with TNF-α stimulated the expression of large T antigen and viral capsid protein-1 mRNA and proteins and BKPyV promoter activity. Knockdown of TNFR1 or TNFR2 expression caused a reduction in TNF-α-stimulated viral replication. NF-κB activation induced by overexpression of constitutively active IKK2 significantly increased viral replication and the activity of the BKPyV promoter containing an NF-κB binding site. The addition of a NF-κB inhibitor on BKPyV-infected cells suppressed viral replication. Blockade of TNF-α functionality by etanercept reduced BKPyV-stimulated expression of TNF-α, interleukin-1ß (IL-1ß), IL-6 and IL-8 and suppressed TNF-α-stimulated viral replication. In cultured HRPTECs and THP-1 cells, BKPyV infection led to increased expression of TNF-α, interleukin-1 ß (IL-1ß), IL-6 and TNFR1 and TNFR2 but the stimulated magnitude was far less than that induced by poly(I:C). This may suggest that BKPyV-mediated autocrine effect is not a major source of TNFα. CONCLUSION: TNF-α stimulates BKPyV replication and inhibition of its signal cascade or functionality attenuates its stimulatory effect. Our study provides a therapeutic anti-BKPyV target.

[Functional Study of TNFR2 Signaling and Drug Discovery Using a Protein Engineering Approach].

Tumor necrosis factor-α (TNF), a proinflammatory cytokine, is critical to the pathogenesis of various inflammatory diseases. There are two subtypes of receptors for TNF, namely type I TNF receptor (TNFR1) and type II TNF receptor (TNFR2). Previous studies using animal models of diseases have demonstrated the predominant role of TNFR1 in the pathogenesis of inflammation. It has recently been proposed that TNFR2 is associated with anti-inflammatory function. This intriguing function of TNFR2 has implications from an immunological and pharmacological perspective. However, the mechanism of the TNFR2-mediated anti-inflammatory effect is not fully understood. In this context, we attempted to elucidate the TNFR2-mediated anti-inflammatory effect and other unknown biological functions of TNFR2 by utilizing our protein engineering technology to generate functional mutant cytokines. Our findings reveal the following. (1) TNFR2 is expressed on regulatory T cells (Tregs) but not conventional T cells (Tconvs) and TNFR2-mediated signals promote proliferation and activation of Tregs. (2) The crystal structure of TNF/TNFR2 complex was solved, which suggests a possible signal initiation mechanism via TNF/TNFR2 cluster formation on the cellular membrane. (3) A novel TNFR2-mediated signal molecule, aminopeptidase P3 (APP3/XPNPEP3), was identified that interacts with TNFR2 as an intracellular adaptor protein. APP3 is required for c-Jun N-terminal kinase (JNK) phosphorylation, the downstream molecule of TNFR2 signal transduction. These results are key to understanding the mechanism of immune regulation and will assist in the identification of immunomodulatory drugs targeting the TNFR2 signaling cascade as well as the function of Tregs.

MiR-125b-5p modulates the function of regulatory T cells in tumor microenvironment by targeting TNFR2.

BACKGROUND: Tumor necrosis factor receptor type 2 (TNFR2) is primarily expressed by CD4+FoxP3+ regulatory T cells (Tregs), especially those present in tumor microenvironment. There is compelling evidence that TNFR2 plays a crucial role in the activation, expansion, and phenotypic stability of Tregs and promotes tumor immune evasion. Understanding of epigenetic regulation of TNFR2 expression in Tregs may help device a novel strategy in cancer immunotherapy. METHODS: MiR-125b-5p-overexpressing or knockdown murine CD4 T cells and Tregs were constructed, and the effect of miR-125b-5p on Tregs proliferation, suppressive function and TNFR2 expression were examined. In vivo antitumor efficacy of Ago-125b-5p (miR-125b-5p agomir) was evaluated in MC38 tumor bearing mice, and tumor-infiltrating Tregs and CD8+ cytotoxic T lymphocytes (CTLs) were analyzed. RNA-seq analysis was applied to reveal the genes and signaling pathways regulated by miR-125b-5p in Tregs. RESULTS: In this study, we found that TNFR2 was a direct target of miR-125b-5p. Overexpression of miR-125b-5p decreased the proportion of Tregs and their expression of TNFR2 and consequently inhibited its proliferation and suppressive function by regulating the metabolism-related signaling pathways. Moreover, in colon cancer bearing mice, the administration of Ago-125b-5p markedly inhibited the tumor growth, which was associated with reduction of Tregs and increase of IFNγ+CD8+ T cells in tumor environment. Furthermore, in human colon adenocarcinoma patients, we verified that miR-125b-5p expression was downregulated, and low levels of miR-125b-5p were associated with poor prognosis. Interestingly, the expression of miR-125b-5p and TNFR2 were negatively correlated. CONCLUSIONS: Our study for the first time found that the expression of TNFR2 by Tregs was regulated by miR-125b-5p. Our results showed that miR-125b-5p had the capacity to inhibit the expression of TNFR2 and immunosuppressive activity of Tregs and consequently enhanced the antitumor efficacy. This property of miR-125b-5p may be therapeutically harnessed in the treatment of human cancers.

Genetic and immune changes in Tibetan high-altitude populations contribute to biological adaptation to hypoxia.

BACKGROUND: Tibetans have lived at very high altitudes for thousands of years, and have a distinctive suite of physiological traits that enable them to tolerate environmental hypoxia. Expanding awareness and knowledge of the differences in hematology, hypoxia-associated genes, immune system of people living at different altitudes and from different ethnic groups may provide evidence for the prevention of mountain sickness. METHOD: Ninety-five Han people at mid-altitude, ninety-five Tibetan people at high-altitude and ninety-eight Han people at high-altitude were recruited. Red blood cell parameters, immune cells, the contents of cytokines, hypoxia-associated gene single nucleotide polymorphisms (SNPs) were measured. RESULTS: The values of Hematocrit (HCT), Mean cell volume (MCV) and Mean cell hemoglobin (MCH) in red blood cell, immune cell CD19+ B cell number, the levels of cytokines Erb-B2 receptor tyrosine kinase 3 (ErbB3) and Tumor necrosis factor receptor II (TNF-RII) and the levels of hypoxia-associated factors Hypoxia inducible factor-1α (HIF-1α), Hypoxia inducible factor-2α (HIF-2α) and HIF prolyl 4-hydroxylase 2 (PHD2) were decreased, while the frequencies of SNPs in twenty-six Endothelial PAS domain protein 1 (EPAS1) and Egl-9 family hypoxia inducible factor 1 (EGLN1) were increased in Tibetan people at high-altitude compared with that of Han peoples at high-altitude. Furthermore, compared with mid-altitude individuals, high-altitude individuals showed lower blood cell parameters including Hemoglobin concentration (HGB), HCT, MCV and MCH, higher Mean cell hemoglobin concentration (MCHC), lower immune cells including CD19+ B cells, CD4+ T cells and CD4/CD8 ratio, higher immune cells containing CD8+ T cells and CD16/56NK cells, decreased Growth regulated oncogene alpha (GROa), Macrophage inflammatory protein 1 beta (MIP-1b), Interleukin-8 (IL-8), and increased Thrombomodulin, downregulated hypoxia-associated factors including HIF1α, HIF2α and PHD2, and higher frequency of EGLN1 rs2275279. CONCLUSIONS: These results indicated that biological adaption to hypoxia at high altitude might have been mediated by changes in immune cells, cytokines, and hypoxia-associated genes during the evolutionary history of Tibetan populations. Furthermore, different responses to high altitude were observed in different ethnic groups, which may provide a useful knowledge to improve the protection of high-altitude populations from mountain sickness.

Downregulation of TNFR2 decreases survival gene expression, promotes apoptosis and affects the cell cycle of gastric cancer cells.

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.