RESUMO
Tumor necrosis factor-α (TNF-α) is a pleiotropic, proinflammatory cytokine related to different neurodegenerative diseases, including Alzheimer's disease (AD). Although the linkage between increased TNF-α levels and AD is widely recognized, TNF-α-neutralizing therapies have failed to treat AD. Previous research has associated this with the antithetic functions of the two TNF receptors, TNF receptor 1, associated with inflammation and apoptosis, and TNF receptor 2 (TNFR2), associated with neuroprotection. In our study, we investigated the effects of specifically stimulating TNFR2 with a TNFR2 agonist (NewStar2) in a transgenic Aß-overexpressing mouse model of AD by administering NewStar2 in two different ways: centrally, via implantation of osmotic pumps, or systemically by intraperitoneal injections. We found that both centrally and systemically administered NewStar2 resulted in a drastic reduction in amyloid ß deposition and ß-secretase 1 expression levels. Moreover, activation of TNFR2 increased microglial and astrocytic activation and promoted the uptake and degradation of Aß. Finally, cognitive functions were also improved after NewStar2 treatment. Our results demonstrate that activation of TNFR2 mitigates Aß-induced cognitive deficits and neuropathology in an AD mouse model and indicates that TNFR2 stimulation might be a potential treatment for AD.
Assuntos
Doença de Alzheimer , Cognição , Receptores Tipo II do Fator de Necrose Tumoral , Animais , Camundongos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Cognição/efeitos dos fármacos , Modelos Animais de Doenças , Camundongos Transgênicos , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Receptores Tipo II do Fator de Necrose Tumoral/agonistas , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Inhibition of CD95/Fas activation is currently under clinical investigation as a therapy for glioblastoma multiforme and preclinical studies suggest that disruption of the CD95-CD95L interaction could also be a strategy to treat inflammatory and neurodegenerative disorders. Besides neutralizing anti-CD95L/FasL antibodies, mainly CD95ed-Fc, a dimeric Fc fusion protein of the extracellular domain of CD95 (CD95ed), is used to prevent CD95 activation. In view of the fact that full CD95 activation requires CD95L-induced CD95 trimerization and clustering of the resulting liganded CD95 trimers, we investigated whether fusion proteins of the extracellular domain of CD95 with a higher valency than CD95ed-Fc have an improved CD95L-neutralization capacity. We evaluated an IgG1(N297A)-based tetravalent CD95ed fusion protein which was obtained by replacing the variable domains of IgG1(N297A) with CD95ed (CD95ed-IgG1(N297A)) and a hexavalent variant obtained by fusion of CD95ed with a TNC-Fc(DANA) scaffold (CD95ed-TNC-Fc(DANA)) promoting hexamerization. The established N297A and DANA mutations were used to minimize FcγR binding of the constructs under maintenance of neonatal Fc receptor (FcRn) binding. Size exclusion high-performance liquid chromatography indicated effective assembly of CD95ed-IgG1(N297A). More important, CD95ed-IgG1(N297A) was much more efficient than CD95ed-Fc in protecting cells from cell death induction by human and murine CD95L. Surprisingly, despite its hexavalent structure, CD95ed-TNC-Fc(DANA) displayed an at best minor improvement of the capacity to neutralize CD95L suggesting that besides valency, other factors, such as spatial organization and agility of the CD95ed domains, play also a role in neutralization of CD95L trimers by CD95ed fusion proteins. More studies are now required to evaluate the superior CD95L-neutralizing capacity of CD95ed-IgG1(N297A) in vivo.
RESUMO
CD4+Foxp3+ regulatory T (Treg) cells are central modulators of autoimmune diseases. However, the timing and location of Treg cell-mediated suppression of tissue-specific autoimmunity remain undefined. Here, we addressed these questions by investigating the role of tumor necrosis factor (TNF) receptor 2 (TNFR2) signaling in Treg cells during experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. We found that TNFR2-expressing Treg cells were critical to suppress EAE at peak disease in the central nervous system but had no impact on T cell priming in lymphoid tissues at disease onset. Mechanistically, TNFR2 signaling maintained functional Treg cells with sustained expression of CTLA-4 and Blimp-1, allowing active suppression of pathogenic T cells in the inflamed central nervous system. This late effect of Treg cells was further confirmed by treating mice with TNF and TNFR2 agonists and antagonists. Our findings show that endogenous Treg cells specifically suppress an autoimmune disease by acting in the target tissue during overt inflammation. Moreover, they bring a mechanistic insight to some of the adverse effects of anti-TNF therapy in patients.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Esclerose Múltipla/imunologia , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Linfócitos T Reguladores/imunologia , Animais , Medula Óssea/patologia , Antígeno CTLA-4/metabolismo , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/patologia , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/patologia , Humanos , Camundongos , Camundongos Knockout , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/patologia , Fator 1 de Ligação ao Domínio I Regulador Positivo/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/agonistas , Receptores Tipo II do Fator de Necrose Tumoral/antagonistas & inibidores , Receptores Tipo II do Fator de Necrose Tumoral/genética , Transdução de Sinais/imunologia , Linfócitos T Reguladores/metabolismoRESUMO
MOTIVATION: Clustering enables TNF receptors to stimulate intracellular signaling. The differential soluble ligand-induced clustering behavior of TNF receptor 1 (TNFR1) and TNFR2 was modeled. A structured, rule-based model implemented ligand-independent pre-ligand binding assembly domain (PLAD)-mediated homotypic low affinity interactions of unliganded and liganded TNF receptors. RESULTS: Soluble TNF initiates TNFR1 signaling but not TNFR2 signaling despite receptor binding unless it is secondarily oligomerized. We consider high affinity binding of TNF to signaling-incompetent pre-assembled dimeric TNFR1 and TNFR2 molecules and secondary clustering of liganded dimers to signaling competent ligand-receptor clusters. Published receptor numbers, affinities and measured different activities of clustered receptors validated model simulations for a large range of receptor and ligand concentrations. Different PLAD-PLAD affinities and different activities of receptor clusters explain the observed differences in the TNF receptor stimulating activities of soluble TNF. AVAILABILITY AND IMPLEMENTATION: All scripts and data are in manuscript and supplement at Bioinformatics online. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Receptores Tipo I de Fatores de Necrose Tumoral , Fator de Necrose Tumoral alfa , Biologia , Ligantes , Transdução de SinaisRESUMO
Targeted therapies using biopharmaceuticals are of growing clinical importance in disease treatment. Currently, there are several limitations of protein-based therapeutics (biologicals), including suboptimal biodistribution, lack of stability, and systemic side effects. A promising approach to overcoming these limitations could be a therapeutic cell-loaded 3D construct consisting of a suitable matrix component that harbors producer cells continuously secreting the biological of interest. Here, the recombinant spider silk proteins eADF4(C16), eADF4(C16)-RGD, and eADF4(C16)-RGE have been processed together with HEK293 producer cells stably secreting the highly traceable reporter biological TNFR2-Fc-GpL, a fusion protein consisting of the extracellular domain of TNFR2, the Fc domain of human IgG1, and the luciferase of Gaussia princeps as a reporter domain. eADF4(C16) and eADF4(C16)-RGD hydrogels provide structural and mechanical support, promote HEK293 cell growth, and allow fusion protein production by the latter. Bioink-captured HEK293 producer cells continuously release functional TNFR2-Fc-GpL over 14 days. Thus, the combination of biocompatible, printable spider silk bioinks with drug-producing cells is promising for generating implantable 3D constructs for continuous targeted therapy.
Assuntos
Produtos Biológicos , Aranhas , Animais , Proteínas de Artrópodes/metabolismo , Células HEK293 , Humanos , Hidrogéis , Imunoglobulina G/metabolismo , Oligopeptídeos/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Proteínas Recombinantes/química , Seda/química , Aranhas/metabolismo , Distribuição TecidualRESUMO
Inhibitory targeting of the CD40L-CD40 system is a promising therapeutic option in the field of organ transplantation and is also attractive in the treatment of autoimmune diseases. After early complex results with neutralizing CD40L antibodies, it turned out that lack of Fcγ receptor (FcγR)-binding is the crucial factor for the development of safe inhibitory antibodies targeting CD40L or CD40. Indeed, in recent years, blocking CD40 antibodies not interacting with FcγRs, has proven to be well tolerated in clinical studies and has shown initial clinical efficacy. Stimulation of CD40 is also of considerable therapeutic interest, especially in cancer immunotherapy. CD40 can be robustly activated by genetically engineered variants of soluble CD40L but also by anti-CD40 antibodies. However, the development of CD40L-based agonists is biotechnologically and pharmacokinetically challenging, and anti-CD40 antibodies typically display only strong agonism in complex with FcγRs or upon secondary crosslinking. The latter, however, typically results in poorly developable mixtures of molecule species of varying stoichiometry and FcγR-binding by anti-CD40 antibodies can elicit unwanted side effects such as antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP) of CD40 expressing immune cells. Here, we summarize and compare strategies to overcome the unwanted target cell-destroying activity of anti-CD40-FcγR complexes, especially the use of FcγR type-specific mutants and the FcγR-independent cell surface anchoring of bispecific anti-CD40 fusion proteins. Especially, we discuss the therapeutic potential of these strategies in view of the emerging evidence for the dose-limiting activities of systemic CD40 engagement.
Assuntos
Ligante de CD40 , Receptores de IgG , Receptores de IgG/metabolismo , Ligante de CD40/farmacologia , Antígenos CD40 , Citotoxicidade Celular Dependente de Anticorpos , Anticorpos NeutralizantesRESUMO
Several drugs targeting members of the TNF superfamily or TNF receptor superfamily (TNFRSF) are widely used in medicine or are currently being tested in therapeutic trials. However, their mechanism of action remains poorly understood. Here, we explored the effects of TNFRSF co-stimulation on murine Foxp3+ regulatory T cell (Treg) biology, as they are pivotal modulators of immune responses. We show that engagement of TNFR2, 4-1BB, GITR, and DR3, but not OX40, increases Treg proliferation and survival. Triggering these TNFRSF in Tregs induces similar changes in gene expression patterns, suggesting that they engage common signal transduction pathways. Among them, we identified a major role of canonical NF-κB. Importantly, TNFRSF co-stimulation improves the ability of Tregs to suppress colitis. Our data demonstrate that stimulation of discrete TNFRSF members enhances Treg activation and function through a shared mechanism. Consequently, therapeutic effects of drugs targeting TNFRSF or their ligands may be mediated by their effect on Tregs.
Assuntos
Ativação Linfocitária , NF-kappa B/imunologia , Receptores do Fator de Necrose Tumoral/imunologia , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia , Animais , Camundongos , Camundongos Knockout , NF-kappa B/genética , Receptores do Fator de Necrose Tumoral/genética , Transdução de Sinais/genética , Linfócitos T Reguladores/citologiaRESUMO
Three new naphthylisoquinoline dimers, jozibrevines A-C (1a-c), were isolated from the West African shrub Ancistrocladus abbreviatus, along with the known dimer jozimine A2 (1d). The two molecular moieties of 1a-d are coupled via the sterically constrained 3',3â³-positions of their two naphthalene units, so that the central biaryl linkage is rotationally hindered. With the two outer axes also being chiral, 1a-d possess three consecutive stereogenic axes. The four isolated dimers all have the same constitutions and identical absolute configurations at the four stereogenic centers, but differ by their axial chirality. They belong to the extremely small class of Dioncophyllaceae-type naphthylisoquinoline dimers, i.e., being devoid of oxygen functions at C-6 and bearing the R-configuration at C-3 in their isoquinoline portions. Besides these dimers, the plant produces predominantly typical Ancistrocladaceae-type monomeric compounds, i.e., with the S-configuration at C-3 and an oxygen function at C-6, such as the new ancistrobrevines K (5) and L (6). Furthermore, a new hybrid-type (i.e., mixed Ancistrocladaceae/Dioncophyllaceae-type) alkaloid was identified, named ancistrobrevine M (7), which is 3R-configured and 6-oxygenated. Remarkable was the discovery of its "inverse hybrid-type" counterpart, dioncoline A (8). It is the as yet only known 3S-configured naphthylisoquinoline lacking an O-functionality at C-6. The new jozibrevines A-C (1a-c) exhibited pronounced antiplasmodial activities in the submicromolar range, with 1a being the most potent compound (IC50, 0.012 µM). Furthermore, jozimine A2 (1d) showed cytotoxicity against human colon carcinoma (HT-29), fibrosarcoma (HT1080), and multiple myeloma (MM.1S) cancer cells, displaying IC50 values of 12.0, 9.0, and 5.0 µM, respectively, whereas jozibrevines A (1a) and B (1b) were nontoxic in this concentration range.
Assuntos
Antimaláricos/química , Antimaláricos/farmacologia , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Caryophyllales/química , Isoquinolinas/química , Isoquinolinas/farmacologia , Naftalenos/química , Naftalenos/farmacologia , África Ocidental , Animais , Antiprotozoários/química , Antiprotozoários/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células HT29 , Humanos , Estrutura Molecular , Raízes de Plantas/química , Plasmodium falciparum/efeitos dos fármacosRESUMO
In the present study, LC-HRESIMS-assisted dereplication along with bioactivity-guided isolation led to targeting two brominated oxindole alkaloids (compounds 1 and 2) which probably play a key role in the previously reported antibacterial, antibiofilm, and cytotoxicity of Callyspongia siphonella crude extracts. Both metabolites showed potent antibacterial activity against Gram-positive bacteria, Staphylococcus aureus (minimum inhibitory concentration (MIC) = 8 and 4 µg/mL) and Bacillus subtilis (MIC = 16 and 4 µg/mL), respectively. Furthermore, they displayed moderate biofilm inhibitory activity in Pseudomonas aeruginosa (49.32% and 41.76% inhibition, respectively), and moderate in vitro antitrypanosomal activity (13.47 and 10.27 µM, respectively). In addition, they revealed a strong cytotoxic effect toward different human cancer cell lines, supposedly through induction of necrosis. This study sheds light on the possible role of these metabolites (compounds 1 and 2) in keeping fouling organisms away from the sponge outer surface, and the possible applications of these defensive molecules in the development of new anti-infective agents.
Assuntos
Alcaloides/farmacologia , Callyspongia/química , Oxindóis/farmacologia , Animais , Anti-Infecciosos/farmacologia , Antiprotozoários/farmacologia , Bactérias/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Linhagem Celular Tumoral , Células HT29 , Halogenação , Humanos , Oceano Índico , Testes de Sensibilidade Microbiana/métodosRESUMO
BACKGROUND AND AIMS: Tumor necrosis factor (TNF) is a cytokine that promotes inflammation and contributes to pathogenesis of inflammatory bowel diseases. Unlike other cells and tissues, intestinal epithelial cells undergo rapid cell death upon exposure to TNF, by unclear mechanisms. We investigated the roles of inhibitor of apoptosis proteins (IAPs) in the regulation of TNF-induced cell death in the intestinal epithelium of mice and intestinal organoids. METHODS: RNA from cell lines and tissues was analyzed by quantitative polymerase chain reaction, protein levels were analyzed by immunoblot assays. BIRC2 (also called cIAP1) was expressed upon induction from lentiviral vectors in young adult mouse colon (YAMC) cells. YAMC cells, the mouse colon carcinoma cell line MC38, the mouse macrophage cell line RAW 264.7, or mouse and human organoids were incubated with second mitochondrial activator of caspases (Smac)-mimetic compound LCL161 or recombinant TNF-like weak inducer of apoptosis (TNFSF12) along with TNF, and cell death was quantified. C57BL/6 mice with disruption of Xiap, Birc2 (encodes cIAP1), Birc3 (encodes cIAP2), Tnfrsf1a, or Tnfrsf1b (Tnfrsf1a and b encode TNF receptors) were injected with TNF or saline (control); liver and intestinal tissues were collected and analyzed for apoptosis induction by cleaved caspase 3 immunohistochemistry. We also measured levels of TNF and alanine aminotransferase in serum from mice. RESULTS: YAMC cells, and mouse and human intestinal organoids, died rapidly in response to TNF. YAMC and intestinal crypts expressed lower levels of XIAP, cIAP1, cIAP2, and cFLIP than liver tissue. Smac-mimetics reduced levels of cIAP1 and XIAP in MC38 and YAMC cells, and Smac-mimetics and TNF-related weak inducer of apoptosis increased TNF-induced cell death in YAMC cells and organoids-most likely by sequestering and degrading cIAP1. Injection of TNF greatly increased levels of cell death in intestinal tissue of cIAP1-null mice, compared with wild-type C57BL/6 mice, cIAP2-null mice, or XIAP-null mice. Excessive TNF-induced cell death in the intestinal epithelium was mediated TNF receptor 1. CONCLUSIONS: In a study of mouse and human cell lines, organoids, and tissues, we found cIAP1 to be required for regulation of TNF-induced intestinal epithelial cell death and survival. These findings have important implications for the pathogenesis of TNF-mediated enteropathies and chronic inflammatory diseases of the intestine.
Assuntos
Apoptose , Células Epiteliais , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteína 3 com Repetições IAP de Baculovírus , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Citocina TWEAK , Células Epiteliais/efeitos dos fármacos , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Fígado/efeitos dos fármacos , Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Organoides , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo II do Fator de Necrose Tumoral/genética , Tiazóis/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Fatores de Necrose Tumoral/farmacologiaRESUMO
A new cyclic hexapeptide, nocardiotide A (1), together with three known compounds-tryptophan (2), kynurenic acid (3), and 4-amino-3-methoxy benzoic acid (4)-were isolated and identified from the broth culture of Nocardiopsis sp. UR67 strain associated with the marine sponge Callyspongia sp. from the Red Sea. The structure elucidation of the isolated compounds were determined based on detailed spectroscopic data including ¹D and ²D nuclear magnetic resonance (NMR) experimental analyses in combination with high resolution electrospray ionization mass spectrometry (HR-ESI-MS), while the absolute stereochemistry of all amino acids components of nocardiotide A (1) was deduced using Marfey's method. Additionally, ten known metabolites were dereplicated using HR-ESI-MS analysis. Nocardiotide A (1) displayed significant cytotoxic effects towards the murine CT26 colon carcinoma, human HeLa cervix carcinoma, and human MM.1S multiple myeloma cell lines. The results obtained revealed sponge-associated Nocardiopsis as a substantial source of lead natural products with pronounced pharmacological activities.
Assuntos
Actinobacteria/química , Antineoplásicos/farmacologia , Callyspongia/microbiologia , Peptídeos Cíclicos/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Organismos Aquáticos/microbiologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Oceano Índico , Camundongos , Neoplasias/tratamento farmacológico , Peptídeos Cíclicos/química , Peptídeos Cíclicos/isolamento & purificaçãoRESUMO
Ligands of the tumor necrosis factor superfamily (TNFSF) interact with members of the TNF receptor superfamily (TNFRSF). TNFSF ligand-TNFRSF receptor interactions have been intensively evaluated by many groups. The affinities of TNFSF ligand-TNFRSF receptor interactions are highly dependent on the oligomerization state of the receptor, and cellular factors (e.g. actin cytoskeleton and lipid rafts) influence the assembly of ligand-receptor complexes, too. Binding studies on TNFSF ligand-TNFRSF receptor interactions were typically performed using cell-free assays with recombinant fusion proteins that contain varying numbers of TNFRSF ectodomains. It is therefore not surprising that affinities determined for an individual TNFSF ligand-TNFRSF interaction differ sometimes by several orders of magnitude and often do not reflect the ligand activity observed in cellular assays. To overcome the intrinsic limitations of cell-free binding studies and usage of recombinant receptor domains, we performed comprehensive binding studies with Gaussia princeps luciferase TNFSF ligand fusion proteins for cell-bound TNFRSF members on intact cells at 37 °C. The affinities of the TNFSF ligand G. princeps luciferase-fusion proteins ranged between 0.01 and 19 nm and offer the currently most comprehensive and best suited panel of affinities for in silico studies of ligand-receptor systems of the TNF family.
Assuntos
Receptores do Fator de Necrose Tumoral/metabolismo , Linhagem Celular Tumoral , Citocinas/metabolismo , Células HEK293 , Humanos , Luciferases/genética , Ligação Proteica , Receptores do Fator de Necrose Tumoral/genética , Proteínas RecombinantesRESUMO
Due to its immunogenicity and overexpression concomitant with leukemia progression, Wilms tumor protein 1 (WT1) is of particular interest for immunotherapy of AML relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT). So far, WT1-specific T-cell responses have mainly been induced by vaccination with peptides presented by certain HLA alleles. However, this approach is still not widely applicable in clinical practice due to common limitations of HLA restriction. Dendritic cell (DC) vaccines electroporated with mRNA encoding full-length protein have also been tested for generating WT1-derived peptides for presentation to T-cells. Alternatively, an efficient and broad WT1 peptide presentation could be elicited by triggering receptor-mediated protein endocytosis of DCs. Therefore, we developed antibody fusion proteins consisting of an antibody specific for the DEC205 endocytic receptor on human DCs and various fragments of WT1 as DC-targeting recombinant WT1 vaccines (anti-hDEC205-WT1). Of all anti-hDEC205-WT1 fusion proteins designed for overcoming insufficient expression, anti-hDEC205-WT110-35, anti-hDEC205-WT191-138, anti-hDEC205-WT1223-273, and anti-hDEC205-WT1324-371 were identified in good yields. The anti-hDEC205-WT191-138 was capable of directly inducing ex vivo T-cell responses by co-incubation of the fusion protein-loaded monocyte-derived mature DCs and autologous T-cells of either healthy or HSCT individuals. Furthermore, the DC-targeted WT191-138-induced specific T-cells showed a strong cytotoxic activity by lysing WT1-overexpressing THP-1 leukemia cells in vitro while sparing WT1-negative hematopoietic cells. In conclusion, our approach identifies four WT1 peptide-antibody fusion proteins with sufficient production and introduces an alternative vaccine that could be easily translated into clinical practice to improve WT1-directed antileukemia immune responses after allo-HSCT.
Assuntos
Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Imunoterapia Adotiva/métodos , Linfócitos T Citotóxicos/imunologia , Proteínas WT1/imunologia , Sequência de Aminoácidos , Animais , Apresentação de Antígeno , Células CHO , Vacinas Anticâncer/genética , Cricetulus , Eletroporação , Células HEK293 , Humanos , Ativação Linfocitária , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Proteínas WT1/genéticaRESUMO
Inhibition of the tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK)/fibroblast growth factor-inducible 14 (Fn14) system reduces intestinal cell death and disease development in several models of colitis. In view of the crucial role of TNF and intestinal cell death in graft-versus-host disease (GVHD) and the ability of TWEAK to enhance TNF-induced cell death, we tested here the therapeutic potential of Fn14 blockade on allogeneic hematopoietic cell transplantation (allo-HCT)-induced intestinal GVHD. An Fn14-specific blocking human immunoglobulin G1 antibody variant with compromised antibody-dependent cellular cytotoxicity (ADCC) activity strongly inhibited the severity of murine allo-HCT-induced GVHD. Treatment of the allo-HCT recipients with this monoclonal antibody reduced cell death of gastrointestinal cells but neither affected organ infiltration by donor T cells nor cytokine production. Fn14 blockade also inhibited intestinal cell death in mice challenged with TNF. This suggests that the protective effect of Fn14 blockade in allo-HCT is based on the protection of intestinal cells from TNF-induced apoptosis and not due to immune suppression. Importantly, Fn14 blockade showed no negative effect on graft-versus-leukemia/lymphoma (GVL) activity. Thus, ADCC-defective Fn14-blocking antibodies are not only possible novel GVL effect-sparing therapeutics for the treatment of GVHD but might also be useful for the treatment of other inflammatory bowel diseases where TNF-induced cell death is of relevance.
Assuntos
Anticorpos Monoclonais Murinos/uso terapêutico , Apoptose , Doença Enxerto-Hospedeiro/prevenção & controle , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Intestinos/patologia , Receptores do Fator de Necrose Tumoral/antagonistas & inibidores , Inibidores do Fator de Necrose Tumoral , Animais , Citotoxicidade Celular Dependente de Anticorpos , Western Blotting , Células Cultivadas , Citocina TWEAK , Modelos Animais de Doenças , Feminino , Imunofluorescência , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/metabolismo , Doença Enxerto-Hospedeiro/patologia , Humanos , Imunoglobulina G/administração & dosagem , Imunoglobulina G/imunologia , Mucosa Intestinal/metabolismo , Intestinos/imunologia , Medições Luminescentes , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores de IgG/imunologia , Receptores de IgG/metabolismo , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/imunologia , Receptores do Fator de Necrose Tumoral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rituximab , Receptor de TWEAK , Fator de Necrose Tumoral alfa/farmacologia , Fatores de Necrose Tumoral/imunologia , Fatores de Necrose Tumoral/metabolismoAssuntos
Transdução de Sinais/efeitos dos fármacos , Ligante Indutor de Apoptose Relacionado a TNF , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose , Humanos , Glicoproteínas de Membrana , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Receptores do Fator de Necrose Tumoral , Fator de Necrose Tumoral alfaRESUMO
Vß5(+) regulatory T cells (Tregs), which are specific for a mouse endogenous retroviral superantigen, become activated and proliferate in response to Friend virus (FV) infection. We previously reported that FV-induced expansion of this Treg subset was dependent on CD8(+) T cells and TNF-α, but independent of IL-2. We now show that the inflammatory milieu associated with FV infection is not necessary for induction of Vß5(+) Treg expansion. Rather, it is the presence of activated CD8(+) T cells that is critical for their expansion. The data indicate that the mechanism involves signaling between the membrane-bound form of TNF-α on activated CD8(+) T cells and TNFR2 on Tregs. CD8(+) T cells expressing membrane-bound TNF-α but no soluble TNF-α remained competent to induce strong Vß5(+) Treg expansion in vivo. In addition, Vß5(+) Tregs expressing only TNFR2 but no TNFR1 were still responsive to expansion. Finally, treatment of naive mice with soluble TNF-α did not induce Vß5(+) Treg expansion, but treatment with a TNFR2-specific agonist did. These results reveal a new mechanism of intercellular communication between activated CD8(+) T cell effectors and Tregs that results in the activation and expansion of a Treg subset that subsequently suppresses CD8(+) T cell functions.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Receptores Tipo II do Fator de Necrose Tumoral/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Animais , Proteínas de Transporte/genética , Feminino , Vírus da Leucemia Murina de Friend/imunologia , Leucemia Experimental/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Tipo I de Fatores de Necrose Tumoral , Receptores Tipo II do Fator de Necrose Tumoral/agonistas , Infecções por Retroviridae/imunologia , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia , Infecções Tumorais por Vírus/imunologiaRESUMO
BACKGROUND & AIMS: Tumor necrosis factor-related apoptosis inducing ligand (TRAIL-R1) (TNFRSF10A) and TRAIL-R2 (TNFRSF10B) on the plasma membrane bind ligands that activate apoptotic and other signaling pathways. Cancer cells also might have TRAIL-R2 in the cytoplasm or nucleus, although little is known about its activities in these locations. We investigated the functions of nuclear TRAIL-R2 in cancer cell lines. METHODS: Proteins that interact with TRAIL-R2 initially were identified in pancreatic cancer cells by immunoprecipitation, mass spectrometry, and immunofluorescence analyses. Findings were validated in colon, renal, lung, and breast cancer cells. Functions of TRAIL-R2 were determined from small interfering RNA knockdown, real-time polymerase chain reaction, Drosha-activity, microRNA array, proliferation, differentiation, and immunoblot experiments. We assessed the effects of TRAIL-R2 overexpression or knockdown in human pancreatic ductal adenocarcinoma (PDAC) cells and their ability to form tumors in mice. We also analyzed levels of TRAIL-R2 in sections of PDACs and non-neoplastic peritumoral ducts from patients. RESULTS: TRAIL-R2 was found to interact with the core microprocessor components Drosha and DGCR8 and the associated regulatory proteins p68, hnRNPA1, NF45, and NF90 in nuclei of PDAC and other tumor cells. Knockdown of TRAIL-R2 increased Drosha-mediated processing of the let-7 microRNA precursor primary let-7 (resulting in increased levels of mature let-7), reduced levels of the let-7 targets (LIN28B and HMGA2), and inhibited cell proliferation. PDAC tissues from patients had higher levels of nuclear TRAIL-R2 than non-neoplastic pancreatic tissue, which correlated with increased nuclear levels of HMGA2 and poor outcomes. Knockdown of TRAIL-R2 in PDAC cells slowed their growth as orthotopic tumors in mice. Reduced nuclear levels of TRAIL-R2 in cultured pancreatic epithelial cells promoted their differentiation. CONCLUSIONS: Nuclear TRAIL-R2 inhibits maturation of the microRNA let-7 in pancreatic cancer cell lines and increases their proliferation. Pancreatic tumor samples have increased levels of nuclear TRAIL-R2, which correlate with poor outcome of patients. These findings indicate that in the nucleus, death receptors can function as tumor promoters and might be therapeutic targets.
Assuntos
Apoptose/fisiologia , Carcinoma Ductal Pancreático/metabolismo , MicroRNAs/metabolismo , Neoplasias Pancreáticas/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias do Colo/metabolismo , Humanos , Neoplasias Renais/metabolismo , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos SCID , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/fisiologiaRESUMO
We found recently that TNF-like weak inducer of apoptosis (TWEAK) and fibroblast growth factor-inducible-14 (Fn14) by virtue of their strong capability to reduce the freely available cytoplasmic pool of TNFR-associated factor (TRAF)2 and cellular inhibitors of apoptosis (cIAPs) antagonize the functions of these molecules in TNFR1 signaling, resulting in sensitization for apoptosis and inhibition of classical NF-κB signaling. In this study, we demonstrate that priming of cells with TWEAK also interferes with activation of the classical NF-κB pathway by CD40. Likewise, there was strong inhibition of CD40 ligand (CD40L)-induced activation of MAPKs in TWEAK-primed cells. FACS analysis and CD40L binding studies revealed unchanged CD40 expression and normal CD40L-CD40 interaction in TWEAK-primed cells. CD40L immunoprecipitates, however, showed severely reduced amounts of CD40 and CD40-associated proteins, indicating impaired formation or reduced stability of CD40L-CD40 signaling complexes. The previously described inhibitory effect of TWEAK on TNFR1 signaling has been traced back to reduced activity of the TNFR1-associated TRAF2-cIAP1/2 ubiquitinase complex and did not affect the stability of the immunoprecipitable TNFR1 receptor complex. Thus, the inhibitory effect of TWEAK on CD40 signaling must be based at least partly on other mechanisms. In line with this, signaling by the CD40-related TRAF2-interacting receptor TNFR2 was also attenuated but still immunoprecipitable in TWEAK-primed cells. Collectively, we show that Fn14 activation by soluble TWEAK impairs CD40L-CD40 signaling complex formation and inhibits CD40 signaling and thus identify the Fn14-TWEAK system as a potential novel regulator of CD40-related cellular functions.
Assuntos
Antígenos CD40/metabolismo , Ligante de CD40/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/fisiologia , Fator 2 Associado a Receptor de TNF/metabolismo , Fatores de Necrose Tumoral/metabolismo , Western Blotting , Antígenos CD40/imunologia , Ligante de CD40/imunologia , Linhagem Celular , Citocina TWEAK , Citometria de Fluxo , Humanos , Imunoprecipitação , Microscopia Confocal , Receptores do Fator de Necrose Tumoral/imunologia , Fator 2 Associado a Receptor de TNF/imunologia , Receptor de TWEAK , Fatores de Necrose Tumoral/imunologiaRESUMO
BACKGROUND: Fn14 is a therapeutic target in various diseases. RESULTS: Anti-Fn14 antibodies activate the alternative NFκB pathway but not other Fn14-related activities induced by soluble or membrane-bound TWEAK. FcγR-bound anti-Fn14 antibodies, however, activate the full spectrum of Fn14-associated activities. CONCLUSION: Anti-Fn14 antibodies elicit agonistic activities differing from those of the natural Fn14 ligand TWEAK. SIGNIFICANCE: These findings influence the rationale of designing Fn14-targeted therapies. The Fn14-specific monoclonal antibodies PDL192 and P4A8, which are under consideration in clinical trials, showed no agonistic activity with respect to IL8 production and cell death induction. However, oligomerization with protein G or binding to Fcγ receptors converted both anti-Fn14 antibodies into potent agonists. TNF-like weak inducer of apoptosis (TWEAK), the ligand of Fn14, occurs naturally in two forms with partly different signaling capabilities, as a membrane-bound ligand and as a soluble trimeric molecule. Although membrane TWEAK strongly triggers all Fn14-associated pathways, soluble TWEAK predominately triggers the alternative nuclear factor κB (NFκB) pathway and enhances TNF-induced cell death but has only a poor effect on the classical NFκB pathway and chemokine production. Thus, the oligomerized and FcγR-bound anti-Fn14 mAbs mimicked the activity of membrane TWEAK. Notably, both anti-Fn14 antibodies significantly triggered p100 processing, the hallmark of the alternative NFκB pathway, and therefore resembled soluble TWEAK. In contrast to the latter, however, the anti-Fn14s showed no effect on TNF receptor 1-induced cell death and P4A8 even blocked the corresponding TWEAK response. Thus, we showed that Fn14 antibodies display an alternative NFκB pathway-specific agonistic activity but fail to phenocopy other activities of soluble TWEAK, whereas oligomerized or FcγR-bound Fn14 antibodies fully mimic the activity of membrane TWEAK. In view of the trivalent nature of the TWEAK-Fn14 interaction, this suggests that the alternative NFκB pathway is uniquely responsive already to Fn14 dimerization enabling antibodies to elicit an unnatural response pattern distinct from that of the naturally occurring Fn14 ligands.