Dominant-negative Tumor Necrosis Factor Research Articles

Inhibition of TNF reduces mechanical orofacial hyperalgesia induced by Complete Freund's Adjuvant by a TRPV1-dependent mechanism in mice.

Inflammation in the orofacial region results in pain and is associated with many pathological states, including migraine, neuralgias and temporomandibular disorder. Although extensively studied, the mechanisms responsible for these conditions are not known and effective treatments are lacking. We reported earlier that the proinflammatory cytokine tumor necrosis factor (TNF) plays an important role in regulation of trigeminal ganglion (TG) neuron function in vitro. In the present study we investigated the role of TNF in mechanical hypersensitivity in mice. We employed the Complete Freund's Adjuvant (CFA)-induced model of orofacial pain and evaluated the effect of blocking of soluble TNF activity by peripheral administration of the novel dominant negative TNF biologic, XPro1595. We show that CFA administration into the lower lip causes hyperalgesia and an increase in both expression of transient receptor potential vanilloid subfamily member 1 (TRPV1) mRNA and in the average intensity of TRPV1 protein immunoreactivity in TG neurons. We also show that intraperitoneal administration of XPro1595 prevents both CFA-induced mechanical hypersensitivity and, as shown in immunohistochemical staining - upregulation of TRPV1 protein expression in TG neurons. We conclude that one of the possible regulatory mechanisms of TNF in pain involves upregulation of the nociceptor TRPV1, and that peripheral treatment with a selective anti-soluble TNF biologic can prevent hyperalgesia caused by inflammation in the orofacial region. Therefore, these new findings suggest that XPro1595 may serve as a novel treatment for orofacial pain disorders.

Toll-like Receptor 4 Mediates Morphine-Induced Neuroinflammation and Tolerance via Soluble Tumor Necrosis Factor Signaling.

Opioid tolerance and the potential for addiction is a significant burden associated with pain management, yet its precise underlying mechanism and prevention remain elusive. Immune signaling contributes to the decreased efficacy of opioids, and we recently demonstrated that Toll-like receptor 4 (TLR4)-mediated neuroinflammation in the periaqueductal gray (PAG) drives tolerance. Tumor necrosis factor (TNF), a product of TLR4 signaling, promotes inflammation and facilitates glutamatergic signaling, key components of opioid tolerance. Therefore, we hypothesize that TLR4-mediated opioid tolerance requires TNF signaling. By expression of a dominant-negative TNF peptide via lentiviral vector injection in rat PAG to sequester soluble TNF (solTNF), we demonstrate that solTNF mediates morphine tolerance induced by TLR4 signaling, stimulates neuroinflammation (increased IL-1β and TLR4 mRNA), and disrupts glutamate reuptake (decreased GLT-1 and GLAST mRNA). We further demonstrate the efficacy of the brain-permeant PEGylated version of the anti-solTNF peptide, XPro1595, injected systemically, to normalize morphine-induced CNS neuroinflammation and morphine- and endotoxin-induced changes in glutamate transport, effectively preserving the efficacy of morphine analgesia and eliminating tolerance. Our findings provide a novel pharmacological target for the prevention of opioid-induced immune signaling, tolerance, and addiction.

AAV-Dominant Negative Tumor Necrosis Factor (DN-TNF) Gene Transfer to the Striatum Does Not Rescue Medium Spiny Neurons in the YAC128 Mouse Model of Huntington's Disease

CNS inflammation is a hallmark of neurodegenerative disease, and recent studies suggest that the inflammatory response may contribute to neuronal demise. In particular, increased tumor necrosis factor (TNF) signaling is implicated in the pathology of both Parkinson's disease (PD) and Alzheimer's disease (AD). We have previously shown that localized gene delivery of dominant negative TNF to the degenerating brain region can limit pathology in animal models of PD and AD. TNF is upregulated in Huntington's disease (HD), like in PD and AD, but it is unknown whether TNF signaling contributes to neuronal degeneration in HD. We used in vivo gene delivery to test whether selective reduction of soluble TNF signaling could attenuate medium spiny neuron (MSN) degeneration in the YAC128 transgenic (TG) mouse model of Huntington's disease (HD). AAV vectors encoding cDNA for dominant-negative tumor necrosis factor (DN-TNF) or GFP (control) were injected into the striatum of young adult wild type WT and YAC128 TG mice and achieved 30–50% target coverage. Expression of dominant negative TNF protein was confirmed immunohistologically and biochemically and was maintained as mice aged to one year, but declined significantly over time. However, the extent of striatal DN-TNF gene transfer achieved in our studies was not sufficient to achieve robust effects on neuroinflammation, rescue degenerating MSNs or improve motor function in treated mice. Our findings suggest that alternative drug delivery strategies should be explored to determine whether greater target coverage by DN-TNF protein might afford some level of neuroprotection against HD-like pathology and/or that soluble TNF signaling may not be the primary driver of striatal neuroinflammation and MSN loss in YAC128 TG mice.

Peripheral administration of the selective inhibitor of soluble tumor necrosis factor (TNF) XPro®1595 attenuates nigral cell loss and glial activation in 6-OHDA hemiparkinsonian rats.

Parkinson's disease (PD) is a complex multi-system age-related neurodegenerative disorder. Targeting the ongoing neuroinflammation in PD patients is one strategy postulated to slow down or halt disease progression. Proof-of-concept studies from our group demonstrated that selective inhibition of soluble Tumor Necrosis Factor (solTNF) by intranigral delivery of dominant negative TNF (DN-TNF) inhibitors reduced neuroinflammation and nigral dopamine (DA) neuron loss in endotoxin and neurotoxin rat models of nigral degeneration. As a next step toward human clinical trials, we aimed to determine the extent to which peripherally administered DN-TNF inhibitor XPro®1595 could: i) cross the blood-brain-barrier in therapeutically relevant concentrations, ii) attenuate neuroinflammation (microglia and astrocyte), and iii) mitigate loss of nigral DA neurons in rats receiving a unilateral 6-hydroxydopamine (6-OHDA) striatal lesion. Rats received unilateral 6-OHDA (20 μg into the right striatum). Three or 14 days after lesion, rats were dosed with XPro®1595 (10 mg/kg in saline, subcutaneous) every third day for 35 days. Forelimb asymmetry was used to assess motor deficits after the lesion; brains were harvested 35 days after the lesion for analysis of XPro®1595 levels, glial activation and nigral DA neuron number. Peripheral subcutaneous dosing of XPro®1595 achieved plasma levels of 1-8 microgram/mL and CSF levels of 1-6 ng/mL depending on the time the rats were killed after final XPro®1595 injection. Irrespective of start date, XPro®1595 significantly reduced microglia and astrocyte number in SNpc whereas loss of nigral DA neurons was attenuated when drug was started 3, but not 14 days after the 6-OHDA lesion. Our data suggest that systemically administered XPro®1595 may have disease-modifying potential in PD patients where inflammation is part of their pathology.

Ceramide sphingolipid signaling mediates Tumor Necrosis Factor (TNF)-dependent toxicity via caspase signaling in dopaminergic neurons.

BackgroundDopaminergic (DA) neurons in the ventral midbrain selectively degenerate in Parkinson’s disease (PD) in part because their oxidative environment in the substantia nigra (SN) may render them vulnerable to neuroinflammatory stimuli. Chronic inhibition of soluble Tumor Necrosis Factor (TNF) with dominant-negative TNF inhibitors protects DA neurons in rat models of parkinsonism, yet the molecular mechanisms and pathway(s) that mediate TNF toxicity remain(s) to be clearly identified. Here we investigated the contribution of ceramide sphingolipid signaling in TNF-dependent toxicity.ResultsCeramide dose-dependently reduced the viability of DA neuroblastoma cells and primary DA neurons and pharmacological inhibition of sphingomyelinases (SMases) with three different inhibitors during TNF treatment afforded significant neuroprotection by attenuating increased endoplasmic reticulum (ER) stress, loss of mitochondrial membrane potential, caspase-3 activation and decreases in Akt phosphorylation. Using lipidomics mass spectrometry we confirmed that TNF treatment not only promotes generation of ceramide, but also leads to accumulation of several atypical deoxy-sphingoid bases (DSBs). Exposure of DA neuroblastoma cells to atypical DSBs in the micromolar range reduced cell viability and inhibited neurite outgrowth and branching in primary DA neurons, suggesting that TNF-induced de novo synthesis of atypical DSBs may be a secondary mechanism involved in mediating its neurotoxicity in DA neurons.ConclusionsWe conclude that TNF/TNFR1-dependent activation of SMases generates ceramide and sphingolipid species that promote degeneration and caspase-dependent cell death of DA neurons. Ceramide and atypical DSBs may represent novel drug targets for development of neuroprotective strategies that can delay or attenuate the progressive loss of nigral DA neurons in patients with PD.

Allergic Lung Inflammation Is Mediated by Soluble Tumor Necrosis Factor (TNF) and Attenuated by Dominant-Negative TNF Biologics

Tumor Necrosis Factor (TNF) is a pleiotropic cytokine consisting of soluble and transmembrane forms, with distinct roles in inflammation and immunity. TNF is an important factor in allergic airway inflammation. However, the disparate functions of soluble (sol) and transmembrane (tm) TNF in lung pathology are not well understood. Our aim was to assess the activities of solTNF and tmTNF in murine models of allergic airway disease, and to evaluate the efficacy of solTNF-selective inhibition. We used ovalbumin sensitization and challenge of TNF knockout, tmTNF knockin, and wild-type C57BL/6 mice to distinguish differences in airway inflammation and hyperreactivity mediated by solTNF and tmTNF. Functions of solTNF and tmTNF in hyperresponsive, wild-type Balb/c mice were assessed by comparing dominant-negative anti-TNF biologics, which antagonize solTNF yet spare tmTNF, to etanercept, a nonselective inhibitor of both TNF forms. Responses in transgenic C57BL/6 mice demonstrated that solTNF, and not tmTNF, is necessary to drive airway inflammation. In Balb/c mice, dominant-negative TNF biologics administered during immunization decreased the recruitment of eosinophils and lymphocytes into the bronchoalveolar space and lung parenchyma, reduced specific serum IgE, goblet-cell hyperplasia, and eosinophilic inflammation, and suppressed methacholine-induced airway hyperreactivity. Concentrations of IL-5, CCL5/RANTES, CCL11/eotaxin, and CCL17/TARC were also reduced in bronchoalveolar lavage. Dominant-negative TNFs reduced lung eosinophilia, even when given only during antigen challenge. The selective inhibition of soluble TNF suppresses inflammation, hyperreactivity, and remodeling in transgenic and wild-type murine models of allergic airway disease, and may offer safety advantages in therapies that preserve the immunoprotective functions of transmembrane TNF.

Delayed Dominant-Negative TNF Gene Therapy Halts Progressive Loss of Nigral Dopaminergic Neurons in a Rat Model of Parkinson's Disease

Parkinson's disease (PD) is a progressive neurodegenerative disorder typified by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). Recent evidence indicates that neuroinflammation may play a critical role in the pathogenesis of PD, particularly tumor necrosis factor (TNF). We have previously shown that soluble TNF (solTNF) is required to mediate robust degeneration induced by 6-hydroxydopamine (6-OHDA) or lipopolysaccharide. What remains unknown is whether TNF inhibition can attenuate the delayed and progressive phase of neurodegeneration. To test this, rats were injected in the SNpc with lentivirus encoding dominant-negative TNF (lenti-DN-TNF) 2 weeks after receiving a 6-OHDA lesion. Remarkably, when examined 5 weeks after the initial 6-OHDA lesion, no further loss of nigral DA neurons was observed. Lenti-DN-TNF also attenuated microglial activation. Together, these data suggest that TNF is likely a critical mediator of nigral DA neuron death during the delayed and progressive phase of neurodegeneration, and that microglia may be the principal cell type involved. These promising findings provide compelling reasons to perform DN-TNF gene transfer studies in nonhuman primates with the long-term goal of using it in the clinic to prevent the delayed and progressive degeneration of DA neurons that gives rise to motor symptoms in PD.

Apoptosis signal-regulating kinase 1 regulates the expression of caspase-11

Caspase-11 is an inducible caspase involved in the regulation of cell death and inflammation. In the present study, we examined whether apoptosis signal-regulating kinase 1 (Ask1)-mediated signaling pathway is involved in the expression of caspase-11 induced by lipopolysaccharide (LPS). We found that the induction of caspase-11 was suppressed by the inhibitors of NADPH oxidase (Nox) or knockdown of Nox4 that acts downstream of toll-like receptor 4 and generates Ask1-activating reactive oxygen species. Overexpression of dominant negative tumor necrosis factor receptor associate factor 6 also suppressed the induction of caspase-11. Importantly, knockdown or dominant negative form of Ask1 suppressed the induction of caspase-11 following LPS stimulation. Taken together, our results show that Ask1 regulates the expression of caspase-11 following LPS stimulation.

Inhibition of soluble TNF signaling in a mouse model of Alzheimer's disease prevents pre-plaque amyloid-associated neuropathology

Microglial activation and overproduction of inflammatory mediators in the central nervous system (CNS) have been implicated in Alzheimer's disease (AD). Elevated levels of the pro-inflammatory cytokine tumor necrosis factor (TNF) have been reported in serum and post-mortem brains of patients with AD, but its role in progression of AD is unclear. Using novel engineered dominant negative TNF inhibitors (DN-TNFs) selective for soluble TNF (solTNF), we investigated whether blocking TNF signaling with chronic infusion of the recombinant DN-TNF XENP345 or a single injection of a lentivirus encoding DN-TNF prevented the acceleration of AD-like pathology induced by chronic systemic inflammation in 3xTgAD mice. We found that chronic inhibition of solTNF signaling with either approach decreased the LPS-induced accumulation of 6E10-immunoreactive protein in hippocampus, cortex, and amygdala. Immunohistological and biochemical approaches using a C-terminal APP antibody indicated that a major fraction of the accumulated protein was likely to be C-terminal APP fragments (β-CTF) while a minor fraction consisted of Aβ40 and 42. Genetic inactivation of TNFR1-mediated TNF signaling in 3xTgAD mice yielded similar results. Taken together, our studies indicate that soluble TNF is a critical mediator of the effects of neuroinflammation on early (pre-plaque) pathology in 3xTgAD mice. Targeted inhibition of solTNF in the CNS may slow the appearance of amyloid-associated pathology, cognitive deficits, and potentially the progressive loss of neurons in AD.

Intranigral Lentiviral Delivery of Dominant-negative TNF Attenuates Neurodegeneration and Behavioral Deficits in Hemiparkinsonian rats

Neuroinflammatory processes have been implicated in the progressive loss of ventral midbrain dopaminergic (DA) neurons that give rise to Parkinson's disease (PD), a late-onset movement disorder that affects 2% of the population over the age of 70 years. We have shown earlier, in two rat models of PD, that inhibition of the proinflammatory cytokine tumor necrosis factor (TNF) through nigral infusion of dominant-negative (DN-TNF) protein (XENP345) attenuates DA neuron loss. The objectives of this study were to develop a constitutive lentiviral vector encoding dominate-negative TNF, and to determine whether a gene therapy approach to deliver DN-TNF directly into the rodent substantia nigra could prevent or attenuate neurotoxin-induced DA neuron loss and associated behavioral deficits. Here we demonstrate that a single injection of lentivirus-expressing DN-TNF into rat substantia nigra, administered concomitant with a striatal 6-hydroxydopamine lesion, results in sufficiently high expression of inhibitor in vivo to attenuate both DA neuron loss and behavioral deficits resulting from striatal dopamine depletion. Our findings demonstrate the feasibility and efficacy of dominant-negative TNF gene transfer as a novel neuroprotective strategy to prevent or delay nigrostriatal pathway degeneration. This strategy holds the potential for therapeutic application in the treatment of PD.

Blocking Soluble Tumor Necrosis Factor Signaling with Dominant-Negative Tumor Necrosis Factor Inhibitor Attenuates Loss of Dopaminergic Neurons in Models of Parkinson's Disease

The mechanisms that trigger or contribute to loss of dopaminergic (DA) neurons in Parkinson's disease (PD) remain unclear and controversial. Elevated levels of tumor necrosis factor (TNF) in CSF and postmortem brains of PD patients and animal models of PD implicate this proinflammatory cytokine in the pathophysiology of the disease; but a role for TNF in mediating loss of DA neurons in PD has not been clearly demonstrated. Here, we report that neutralization of soluble TNF (solTNF) in vivo with the engineered dominant-negative TNF compound XENP345 (a PEGylated version of the TNF variant A145R/I97T) reduced by 50% the retrograde nigral degeneration induced by a striatal injection of the oxidative neurotoxin 6-hydroxydopamine (6-OHDA). XENP345 was neuroprotective only when infused into the nigra, not the striatum. XENP345/6-OHDA rats displayed attenuated amphetamine-induced rotational behavior, indicating preservation of striatal dopamine levels. Similar protective effects were observed with chronic in vivo coinfusion of XENP345 with bacterial lipopolysaccharide (LPS) into the substantia nigra, confirming a role for solTNF-dependent neuroinflammation in nigral degeneration. In embryonic rat midbrain neuron/glia cell cultures exposed to LPS, even delayed administration of XENP345 prevented selective degeneration of DA neurons despite sustained microglia activation and secretion of solTNF. XENP345 also attenuated 6-OHDA-induced DA neuron toxicity in vitro. Collectively, our data demonstrate a role for TNF in vitro and in vivo in two models of PD, and raise the possibility that delaying the progressive degeneration of the nigrostriatal pathway in humans is therapeutically feasible with agents capable of blocking solTNF in early stages of PD.

The role of TNF in in vitro and in vivo models of neurodegeneration

Using our novel engineered dominant negative Tumor Necrosis Factor variants (DN-TNFs) in models of Parkinson’s Disease (PD) and Alzheimer’s Disease (AD), we are identifying TNF-dependent signaling mechanisms that elicit death of DA neurons as well as TNF-dependent microglial activities that exacerbate amyloid beta-associated neurotoxicity and death of cholinergic neurons. In mouse models of AD, peripheral inflammation (LPS) exacerbates amyloid and tau pathology in hippocampus; we investigated whether blocking soluble TNF signaling with the infusion of DN-TNFs could block the progressive neuropathology or ameliorate the LPS effects. Inhibition of TNF signaling in vitro with DN-TNFs attenuates LPS- induced microglia activation and loss of DA neurons; delayed TNF inhibition still rescues DA neurons and provides more robust rescue than anti-oxidant pretreatment. Experiments are underway to investigate which signaling pathways transduce the neuroinflammatory and neurotoxic effects of TNF. In vivo, unilateral intrastriatal injections of 6-OHDA or intranigral chronic low dose LPS infusion result in a 70–80% loss of ipsilateral nigral DA neurons. Co-administration of DN-TNFs on the lesion side reduces neuronal loss by half and attenuates ipsiversive circling behavior. These results suggest that targeting the TNF pathway may be a reasonable therapeutic approach to halt or slow the progressive loss of DA neurons in PD. Funding by MJ Fox Foundation, American Health Assistance Foundation, and The University of Texas Southwestern Medical Center at Dallas Alzheimer’s Disease Center; NIH,NIA P30AG12300.

Computational design of variant TNF molecules: a novel methodology for inhibition of proinflammatory cascades.

Site-directed mutagenesis of tumor necrosis factor (TNF) based on prediction of the interaction of specific residues with TNF receptors generated dominant-negative constructs, in which single- or double-amino acid changes result in decreased receptor binding and cellular activation. These dominant-negatives not only provide a novel manner to block the proinflammatory effects of TNF, but also can be used as a tool to examine ligand-receptor interactions and their importance in signaling. Because these TNF mutant molecules are smaller than those used for conventional anti-TNF therapies, such as etanercept or infliximab, they are likely to achieve greater tissue concentrations and may provide enhanced therapeutic effect. However, the immunogenicity, as well as efficacy, of the dominant-negative TNF constructs must be more completely examined.

Bypassing Inflammatory Signaling

Tumor necrosis factor (TNF) binds to TNF receptors and stimulates inflammatory responses. In patients, an elevated level of TNF is associated with diseases such as rheumatoid arthritis, and inhibition of TNF signaling can be used to treat these diseases. Steed et al. describe an approach that prevents TNF signaling. They used structure-based protein design to engineer dominant-negative TNFs that form heterotrimers with native TNF and in turn prevent binding to TNF receptors. Experiments in animal models show that this approach has promise in attenuating TNF-mediated pathology in vivo. P. M. Steed, M. G. Tansey, J. Zalevsky, E. A. Zhukovsky, J. R. Desjarlais, D. E. Szymkowski, C. Abbott, D. Carmichael, C. Chan, L. Cherry, P. Cheung, A. J. Chirino, H. H. Chung, S. K. Doberstein, A. Eivazi, A. V. Filikov, S. X. Gao, R. S. Hubert, M. Hwang, L. Hyun, S. Kashi, A. Kim, E. Kim, J. Kung, S. P. Martinez, U. S. Muchhal, D.-H. T. Nguyen, C. O'Brien, D. O'Keefe, K. Singer, O. Vafa, J. Vielmetter, S. C. Yoder, B. I. Dahiyat, Inactivation of TNF signaling by rationally designed dominant-negative TNF variants. Science 301 , 1895-1898 (2003). [Abstract] [Full Text]

Inactivation of TNF signaling by rationally designed dominant-negative TNF variants.

Tumor necrosis factor (TNF) is a key regulator of inflammatory responses and has been implicated in many pathological conditions. We used structure-based design to engineer variant TNF proteins that rapidly form heterotrimers with native TNF to give complexes that neither bind to nor stimulate signaling through TNF receptors. Thus, TNF is inactivated by sequestration. Dominant-negative TNFs represent a possible approach to anti-inflammatory biotherapeutics, and experiments in animal models show that the strategy can attenuate TNF-mediated pathology. Similar rational design could be used to engineer inhibitors of additional TNF superfamily cytokines as well as other multimeric ligands.

Micrococci and peptidoglycan activate TLR2-->MyD88-->IRAK-->TRAF-->NIK-->IKK-->NF-kappaB signal transduction pathway that induces transcription of interleukin-8.

This study was done to elucidate the signal transduction pathway of interleukin-8 (IL-8) induction by gram-positive bacteria. Bacteria (micrococci) and peptidoglycan (PGN) induced transcription of IL-8 in HEK293 cells expressing Toll-like receptor 2 (TLR2) and CD14 but not in those expressing TLR1 or TLR4. A mutation within the NF-kappaB site in the IL-8 promoter abrogated transcriptional induction of IL-8 by the two stimulants. Dominant negative myeloid differentiation protein (MyD88), IL-1 receptor-associated kinase (IRAK), NFkappaB-inducing kinase (NIK), and IkappaB kinase (IKK) mutant forms completely inhibited micrococcus- and PGN-induced activation of NF-kappaB and expression of the gene for IL-8. Induction of NF-kappaB was partially inhibited by dominant negative tumor necrosis factor receptor-associated kinase 6 (TRAF6) but not TRAF2, whereas induction of IL-8 gene was partially inhibited by both TRAF6 and TRAF2. These data indicate that micrococci and PGN induce TLR2-dependent activation of the gene for IL-8 and that this activation requires MyD88, IRAK, NIK, IKK, and NF-kappaB and may also utilize TRAF6 and, to a lesser extent, TRAF2.

UVB-induced association of tumor necrosis factor (TNF) receptor 1/TNF receptor-associated factor-2 mediates activation of Rel proteins.

Exposure of mammalian skin to UV light results in induced gene transcription, playing a role in inflammation, immunosuppression, and tumor promotion. One important group of transcription factors induced by UV radiation is composed of members of the Rel/NF-kappa B family, which are known to play a major role in the transcriptional activation of many genes encoding inflammatory cytokines, adhesion molecules, and viral proteins. However, the upstream events in the transduction of the UVB signal to Rel protein activity are, as yet, unknown. Here, we provide biochemical evidence that exposure of keratinocytes to UVB causes rapid association of tumor necrosis factor (TNF) receptor 1 with its downstream partner TRAF-2. The functional relevance of this association is demonstrated by experiments showing that expression of a dominant negative TNF receptor 1 or TRAF-2 protein inhibits UVB-induced Rel-dependent transcription. Inclusion of a neutralizing antibody toward TNF alpha has no effect on UVB activation of a Rel-responsive reporter gene. Therefore, UVB-induced activation of Rel proteins via TNF receptor 1, independent of ligand activation, is a key component in the UV response in keratinocytes.