Pathogenic TNF-α drives peripheral nerve inflammation in an Aire-deficient model of autoimmunity.

Immune cells infiltrate the peripheral nervous system (PNS) after injury and with autoimmunity, but their net effect is divergent. After injury, immune cells are reparative, while in inflammatory neuropathies (e.g., Guillain Barré Syndrome and chronic inflammatory demyelinating polyneuropathy), immune cells are proinflammatory and promote autoimmune demyelination. An understanding of immune cell phenotypes that distinguish these conditions may, therefore, reveal new therapeutic targets for switching immune cells from an inflammatory role to a reparative state. In an autoimmune regulator (Aire)-deficient mouse model of inflammatory neuropathy, we used single-cell RNA sequencing of sciatic nerves to discover a transcriptionally heterogeneous cellular landscape, including multiple myeloid, innate lymphoid, and lymphoid cell types. Analysis of cell-cell ligand-receptor interactions uncovered a macrophage-mediated tumor necrosis factor-α (TNF-α) signaling axis that is induced by interferon-γ and required for initiation of autoimmune demyelination. Developmental trajectory visualization suggested that TNF-α signaling is associated with metabolic reprogramming of macrophages and polarization of macrophages from a reparative state in injury to a pathogenic, inflammatory state in autoimmunity. Autocrine TNF-α signaling induced macrophage expression of multiple genes (Clec4e, Marcksl1, Cxcl1, and Cxcl10) important in immune cell activation and recruitment. Genetic and antibody-based blockade of TNF-α/TNF-α signaling ameliorated clinical neuropathy, peripheral nerve infiltration, and demyelination, which provides preclinical evidence that the TNF-α axis may be effectively targeted to resolve inflammatory neuropathies.

TWEAK/Fn14 Signaling Involvement in the Pathogenesis of Cutaneous Disease in the MRL/lpr Model of Spontaneous Lupus.

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK, TNFSF12) and its sole receptor Fn14, belonging to the TNF ligand and receptor superfamilies respectively, are involved in cell survival and cytokine production. The role of TWEAK/Fn14 interactions in the pathogenesis of cutaneous lupus has not been explored. TWEAK treatment of murine PAM212 keratinocytes stimulated the secretion of RANTES via Fn14 and promoted apoptosis. Parthenolide, but not wortmanin or the MAPK inhibitor PD98059, significantly decreased production of RANTES, indicating that this effect of TWEAK is mediated via NF-κB signaling. UVB irradiation significantly upregulated the expression of Fn14 on keratinocytes in vitro and in vivo and increased RANTES production. MRL/lpr Fn14 knockout (KO) lupus mice were compared with MRL/lpr Fn14 wild-type (WT) mice to evaluate for any possible differences in the severity of cutaneous lesions and the presence of infiltrating immune cells. MRL/lpr Fn14 KO mice had markedly attenuated cutaneous disease as compared with their Fn14 WT littermates, as evidenced by the well-maintained architecture of the skin and significantly decreased skin infiltration of T cells and macrophages. Our data strongly implicate TWEAK/Fn14 signaling in the pathogenesis of the cutaneous manifestations in the MRL/lpr model of spontaneous lupus and suggest a possible target for therapeutic intervention.

Role of Fn14 in acute alcoholic steatohepatitis in mice.

TNF-like weak inducer of apoptosis (TWEAK) is a growth factor for bipotent liver progenitors that express its receptor, fibroblast growth factor-inducible 14 (Fn14), a TNF receptor superfamily member. Accumulation of Fn14(+) progenitors occurs in severe acute alcoholic steatohepatitis (ASH) and correlates with acute mortality. In patients with severe ASH, inhibition of TNF-α increases acute mortality. The aim of this study was to determine whether deletion of Fn14 improves the outcome of liver injury in alcohol-consuming mice. Wild-type (WT) and Fn14 knockout (KO) mice were fed control high-fat Lieber deCarli diet or high-fat Lieber deCarli diet with 2% alcohol (ETOH) and injected intraperitoneally with CCl4 for 2 wk to induce liver injury. Mice were euthanized 3 or 10 days after CCl4 treatment. Survival was assessed. Liver tissues were analyzed for cell death, inflammation, proliferation, progenitor accumulation, and fibrosis by quantitative RT-PCR, immunoblot, hydroxyproline content, and quantitative immunohistochemistry. During liver injury, Fn14 expression, apoptosis, inflammation, hepatocyte replication, progenitor and myofibroblast accumulation, and fibrosis increased in WT mice fed either diet. Mice fed either diet expressed similar TWEAK/Fn14 levels, but ETOH-fed mice had higher TNF-α expression. The ETOH-fed group developed more apoptosis, inflammation, fibrosis, and regenerative responses. Fn14 deletion did not reduce hepatic TNF-α expression but improved all injury parameters in mice fed the control diet. In ETOH-fed mice, Fn14 deletion inhibited TNF-α induction and increased acute mortality, despite improvement in liver injury. Fn14 mediates wound-healing responses that are necessary to survive acute liver injury during alcohol exposure.

Inflammasome-dependent and -independent IL-18 production mediates immunity to the ISCOMATRIX adjuvant.

Adjuvants are an essential component of modern vaccines and used for their ability to elicit immunity to coadministered Ags. Many adjuvants in clinical development are particulates, but how they drive innate and adaptive immune responses remains poorly understood. Studies have shown that a number of vaccine adjuvants activate inflammasome pathways in isolated APCs. However, the contribution of inflammasome activation to vaccine-mediated immunity in vivo remains controversial. In this study, we evaluated immune cell responses to the ISCOMATRIX adjuvant (IMX) in mice. Like other particulate vaccine adjuvants, IMX potently activated the NALP-3-ASC-Caspase-1 inflammasome in APCs, leading to IL-1ß and IL-18 production. The IL-18R pathway, but not IL-1R, was required for early innate and subsequent cellular immune responses to a model IMX vaccine. APCs directly exposed to IMX underwent an endosome-mediated cell-death response, which we propose initiates inflammatory events locally at the injection site. Importantly, both inflammasome-related and -unrelated pathways contributed to IL-18 dependence in vivo following IMX administration. TNF-α provided a physiological priming signal for inflammasome-dependent IL-18 production by APCs, which correlated with reduced vaccine-mediated immune cell responses in TNF-α- or TNFR-deficient mice. Taken together, our findings highlight an important disconnect between the mechanisms of vaccine adjuvant action in vitro versus in vivo.

Attenuated thermoregulatory, metabolic, and liver acute phase protein response to heat stroke in TNF receptor knockout mice.

Tumor necrosis factor (TNF) is considered an adverse mediator of heat stroke (HS) based on clinical studies showing high serum levels. However, soluble TNF receptors (sTNFR; TNF antagonists) were higher in survivors than nonsurvivors, and TNFR knockout (KO) mice showed a trend toward increased mortality, suggesting TNF has protective actions for recovery. We delineated TNF actions in HS by comparing thermoregulatory, metabolic, and inflammatory responses between B6129F2 (wild type, WT) and TNFR KO mice. Before heat exposure, TNFR KO mice showed ~0.4°C lower core temperature (T(c); radiotelemetry), ~10% lower metabolic rate (M(r); indirect calorimetry), and reduced plasma interleukin (IL)-1α and sIL-1RI than WT mice. KO mice selected warmer temperatures than WT mice in a gradient but remained hypothermic. In the calorimeter, both genotypes showed a similar heating rate, but TNFR KO maintained lower T(c) and M(r) than WT mice for a given heat exposure duration and required ~30 min longer to reach maximum T(c) (42.4°C). Plasma IL-6 increased at ~3 h of recovery in both genotypes, but KO mice showed a more robust sIL-6R response. Higher sIL-6R in the KO mice was associated with delayed liver p-STAT3 protein expression and attenuated serum amyloid A3 (SAA3) gene expression, suggesting the acute phase response (APR) was attenuated in these mice. Our data suggest that the absence of TNF signaling induced a regulated hypothermic state in the KO mice, TNF-IL-1 interactions may modulate T(c) and M(r) during homeostatic conditions, and TNF modulates the APR during HS recovery through interactions with the liver IL-6-STAT3 pathway of SAA3 regulation.

Death receptor 6 regulates adult experience-dependent cortical plasticity.

Sensory experience alters cortical circuitry by parallel processes of axon outgrowth and pruning, but the mechanisms that control these rearrangements are poorly understood. Using in vivo 2-photon longitudinal imaging, we found a marked reduction in axonal pruning in somatosensory cortex of mice with a knock-out of the DR6 gene, which codes for Death Receptor 6. This effect was seen for both long-range horizontal excitatory connections and for the axons of inhibitory neurons. These results identify a new pathway governing axonal plasticity associated with experience-dependent changes in cortical maps.

TNF-like weak inducer of apoptosis (TWEAK) promotes beta cell neogenesis from pancreatic ductal epithelium in adult mice.

AIM/HYPOTHESIS: The adult mammalian pancreas has limited ability to regenerate in order to restore adequate insulin production from multipotent progenitors, the identity and function of which remain poorly understood. Here we test whether the TNF family member TWEAK (TNF-like weak inducer of apoptosis) promotes ß-cell neogenesis from proliferating pancreatic ductal epithelium in adult mice. METHODS: C57Bl/6J mice were treated with Fc-TWEAK and pancreas harvested at different time points for analysis by histology and immunohistochemistry. For lineage tracing, 4 week old double transgenic mice CAII-CreER(TM): R26R-eYFP were implanted with tamoxifen pellet, injected with Fc-TWEAK or control Ig twice weekly and analyzed at day 18 for TWEAK-induced duct cell progeny by costaining for insulin and YFP. The effect of TWEAK on pancreatic regeneration was determined by pancytokeratin immunostaining of paraffin embedded sections from wildtype and TWEAK receptor (Fn14) deficient mice after Px. RESULTS: TWEAK stimulates proliferation of ductal epithelial cells through its receptor Fn14, while it has no mitogenic effect on pancreatic α- or ß-cells or acinar cells. Importantly, TWEAK induces transient expression of endogenous Ngn3, a master regulator of endocrine cell development, and induces focal ductal structures with characteristics of regeneration foci. In addition, we identify by lineage tracing TWEAK-induced pancreatic ß-cells derived from pancreatic duct epithelial cells. Conversely, we show that Fn14 deficiency delays formation of regenerating foci after Px and limits their expansion. CONCLUSIONS/INTERPRETATION: We conclude that TWEAK is a novel factor mediating pancreatic ß-cell neogenesis from ductal epithelium in normal adult mice.

Galectin-9 protein is up-regulated in astrocytes by tumor necrosis factor and promotes encephalitogenic T-cell apoptosis.

Demyelination and axonal damage in multiple sclerosis (MS) are thought to be a consequence of inflammatory processes that are perpetuated by activated glia and infiltrating leukocytes. Galectin-9 is a ß-galactoside binding lectin capable of modulating immune responses and appears to be up-regulated in MS. However, its role in the pathogenesis of MS has yet to be determined. Here, we report that proinflammatory cytokines induce galectin-9 (Gal-9) expression in primary astrocytes and the mechanism by which TNF up-regulates Gal-9. Astrocytes did not express Gal-9 under basal conditions nor did IL-6, IL-10, or IL-13 trigger Gal-9 expression. In contrast, IL-1ß, IFN-γ, and particularly TNF up-regulated Gal-9 in astrocytes. TNF-induced Gal-9 expression was dependent on TNF receptor 1 (TNFR1) as TNF failed to induce Gal-9 in TNFR1(-/-) astrocytes. Blockade of the JNK MAP kinase pathway with the JNK inhibitor SP600125 abrogated TNF-induced Gal-9, whereas p38 and MEK inhibitors had minimal effects. Furthermore, specific knockdown of c-Jun via siRNA in astrocytes before TNF treatment greatly suppressed Gal-9 transcription, suggesting that TNF induces astroglial Gal-9 through the TNF/TNFR1/JNK/cJun signaling pathway. Finally, utilizing astrocytes from Lgals9 mutant (Gal-9(-/-)) mice as well as a myelin basic protein-specific Tim-3(+) encephalitogenic T-cell clone (LCN-8), we found that conditioned medium from TNF-stimulated Gal-9(+/+) but not Gal-9(-/-) astrocytes increased the percentage of apoptotic encephalitogenic T-cells. Together, our results suggest that Gal-9 is induced in astrocytes by TNF via the JNK/c-Jun pathway and that astrocyte-derived Gal-9 may function as an immunoregulatory protein in response to ongoing neuroinflammation.

Neuropsychiatric disease in murine lupus is dependent on the TWEAK/Fn14 pathway.

Given the early onset of neuropsychiatric disease and the potential response to immunosuppressive therapy, neuropsychiatric disease is considered a primary disease manifestation in systemic lupus erythematosus (SLE). However, the pathogenesis is not fully understood and optimal treatment has yet to be determined. TWEAK is a TNF family ligand that mediates pleotropic effects through its receptor Fn14, including the stimulation of inflammatory cytokine production by astrocytes, endothelial cells, and other non-hematopeotic cell types, and induction of neuronal death. Furthermore, TWEAK-inducible mediators are implicated in neuropsychiatric lupus. Thus, we hypothesized that the TWEAK/Fn14 pathway may be involved in the pathogenesis of neuropsychiatric SLE. We generated MRL-lpr/lpr (MRL/lpr) mice deficient for Fn14, the sole known signaling receptor for TWEAK. Neuropsychiatric disease was compared in age- and gender-matched MRL/lpr Fn14 wild type (WT) and knockout (KO) mice, using a comprehensive battery of neurobehavioral tests. We found that MRL/lpr Fn14WT mice displayed profound depression-like behavior as seen by increased immobility in a forced swim test and loss of preference for sweetened fluids, which were significantly ameliorated in Fn14KO mice. Similarly, MRL/lpr Fn14WT mice had impaired cognition, and this was significantly improved in Fn14KO mice. To determine the mechanism by which Fn14 deficiency ameliorates neuropsychiatric disease, we assessed the serum levels of autoantibodies and local expression of cytokines in the cortex and hippocampus of lupus mice. No significant differences were found in the serum levels of antibodies to nuclear antigens, or autoantibodies specifically associated with neuropsychiatric disease, between MRL/lpr Fn14WT and KO mice. However, MRL/lpr Fn14KO mice had significantly decreased brain expression of RANTES, C3, and other proinflammatory mediators. Furthermore, MRL/lpr Fn14KO mice displayed improved blood brain barrier integrity. In conclusion, several central manifestations of neuropsychiatric lupus, including depression-like behavior and altered cognition, are normalized in MRL/lpr mice lacking Fn14. Our results are the first to indicate a role for the TWEAK/Fn14 pathway in the pathogenesis of neuropsychiatric lupus, and suggest this ligand-receptor pair as a potential therapeutic target for a common and dangerous disease manifestation.

A DR6/p75(NTR) complex is responsible for ß-amyloid-induced cortical neuron death.

The p75 neurotrophin receptor (p75(NTR)) is a known mediator of ß-amyloid (Aß)-induced neurotoxicity implicated in Alzheimer's disease (AD). Here, we demonstrate that death receptor 6 (DR6) binds to p75(NTR) and is a component of the p75(NTR) signaling complex responsible for Aß-induced cortical neuron death. Cortical neurons isolated from either DR6 or p75(NTR) null mice are resistant to Aß-induced neurotoxicity. Blocking DR6 function in cortical neurons by anti-DR6 antibodies that block the binding of DR6 to p75(NTR) receptor complex or by a dominant negative DR6 construct lacking the cytoplasmic signaling death domain attenuates Aß-induced caspase 3 activation and cell death. DR6 expression is upregulated in AD cortex and correlates with elevated neuronal death. Targeting the disruption of the DR6/p75(NTR) complex to prevent Aß cytotoxicity represents a new approach for the treatment of neurodegenerative disorders such as AD.

Hepatic macrophages but not dendritic cells contribute to liver fibrosis by promoting the survival of activated hepatic stellate cells in mice.

UNLABELLED: Although it is well established that hepatic macrophages play a crucial role in the development of liver fibrosis, the underlying mechanisms remain largely elusive. Moreover, it is not known whether other mononuclear phagocytes such as dendritic cells (DCs) contribute to hepatic stellate cell (HSC) activation and liver fibrosis. We show for the first time that hepatic macrophages enhance myofibroblast survival in a nuclear factor kappa B (NF-κB)-dependent manner and thereby promote liver fibrosis. Microarray and pathway analysis revealed no induction of HSC activation pathways by hepatic macrophages but a profound activation of the NF-κB pathway in HSCs. Conversely, depletion of mononuclear phagocytes during fibrogenesis in vivo resulted in suppressed NF-κB activation in HSCs. Macrophage-induced activation of NF-κB in HSCs in vitro and in vivo was mediated by interleukin (IL)-1 and tumor necrosis factor (TNF). Notably, IL-1 and TNF did not promote HSC activation but promoted survival of activated HSCs in vitro and in vivo and thereby increased liver fibrosis, as demonstrated by neutralization in coculture experiments and genetic ablation of IL-1 and TNF receptor in vivo. Coculture and in vivo ablation experiments revealed only a minor contribution to NF-κB activation in HSCs by DCs, and no contribution of DCs to liver fibrosis development, respectively. CONCLUSION: Promotion of NF-κB-dependent myofibroblast survival by macrophages but not DCs provides a novel link between inflammation and fibrosis.

Chronic neuron- and age-selective down-regulation of TNF receptor expression in triple-transgenic Alzheimer disease mice leads to significant modulation of amyloid- and Tau-related pathologies.

Neuroinflammation, through production of proinflammatory molecules and activated glial cells, is implicated in Alzheimer's disease (AD) pathogenesis. One such proinflammatory mediator is tumor necrosis factor α (TNF-α), a multifunctional cytokine produced in excess and associated with amyloid ß-driven inflammation and cognitive decline. Long-term global inhibition of TNF receptor type I (TNF-RI) and TNF-RII signaling without cell or stage specificity in triple-transgenic AD mice exacerbates hallmark amyloid and neurofibrillary tangle pathology. These observations revealed that long-term pan anti-TNF-α inhibition accelerates disease, cautions against long-term use of anti-TNF-α therapeutics for AD, and urges more selective regulation of TNF signaling. We used adeno-associated virus vector-delivered siRNAs to selectively knock down neuronal TNF-R signaling. We demonstrate divergent roles for neuronal TNF-RI and TNF-RII where loss of opposing TNF-RII leads to TNF-RI-mediated exacerbation of amyloid ß and Tau pathology in aged triple-transgenic AD mice. Dampening of TNF-RII or TNF-RI+RII leads to a stage-independent increase in Iba-1-positive microglial staining, implying that neuronal TNF-RII may act nonautonomously on the microglial cell population. These results reveal that TNF-R signaling is complex, and it is unlikely that all cells and both receptors will respond positively to broad anti-TNF-α treatments at various stages of disease. In aggregate, these data further support the development of cell-, stage-, and/or receptor-specific anti-TNF-α therapeutics for AD.

Cytotoxic effector function of CD4-independent, CD8(+) T cells is mediated by TNF-α/TNFR.

BACKGROUND: Liver parenchymal cell allografts initiate both CD4-dependent and CD4-independent, CD8(+) T cell-mediated acute rejection pathways. The magnitude of allospecific CD8(+) T cell in vivo cytotoxic effector function is maximal when primed in the presence of CD4(+) T cells. The current studies were conducted to determine if and how CD4(+) T cells might influence cytotoxic effector mechanisms. METHODS: Mice were transplanted with allogeneic hepatocytes. In vivo cytotoxicity assays and various gene-deficient recipient mice and target cells were used to determine the development of Fas-, TNF-α-, and perforin-dependent cytotoxic effector mechanisms after transplantation. RESULTS: CD8(+) T cells maturing in CD4-sufficient hepatocyte recipients develop multiple (Fas-, TNF-α-, and perforin-mediated) cytotoxic mechanisms. However, CD8(+) T cells, maturing in the absence of CD4(+) T cells, mediate cytotoxicity and transplant rejection that is exclusively TNF-α/TNFR-dependent. To determine the kinetics of CD4-mediated help, CD4(+) T cells were adoptively transferred into CD4-deficient mice at various times posttransplant. The maximal influence of CD4(+) T cells on the magnitude of CD8-mediated in vivo allocytotoxicityf occurs within 48 hours. CONCLUSION: The implication of these studies is that interference of CD4(+) T cell function by disease or immunotherapy will have downstream consequences on both the magnitude of allocytotoxicity as well as the cytotoxic effector mechanisms used by allospecific CD8(+) cytolytic T cells.

Inhibition of the TWEAK/Fn14 pathway attenuates renal disease in nephrotoxic serum nephritis.

Previously it was shown that the TNF superfamily member TWEAK (TNFSF12) acts through its receptor, Fn14, to promote proinflammatory responses in kidney cells, including the production of MCP-1, RANTES, IP-10 and KC. In addition, the TWEAK/Fn14 pathway promotes mesangial cell proliferation, vascular cell activation, and renal cell death. To study the relevance of the TWEAK/Fn14 pathway in the pathogenesis of antibody-induced nephritis using the mouse model of nephrotoxic serum nephritis (NTN), we induced NTN by passive transfer of rabbit anti-glomerular antibodies into Fn14 knockout (KO) and wild type (WT) mice. Severe proteinuria as well as renal histopathology were induced in WT but not in Fn14 KO mice. Similarly, a pharmacologic approach of anti-TWEAK mAb administration into WT mice in the NTN model significantly ameliorated proteinuria and improved kidney histology. Anti-TWEAK treatment did not affect the generation of mouse anti-rabbit antibodies; however, within the kidney there was a significant decrease in glomerular immunoglobulin deposition, as well as macrophage infiltrates and tubulointerstitial fibrosis. The mechanism of action is most likely due to reductions in downstream targets of TWEAK/Fn14 signaling, including reduced renal expression of MCP-1, VCAM-1, IP-10, RANTES as well as Fn14 itself, and other molecular pathways associated with fibrosis in anti-TWEAK treated mice. Thus, TWEAK/Fn14 interactions are instrumental in the pathogenesis of nephritis in the NTN model, apparently mediating a cascade of pathologic events locally in the kidney rather than by impacting the systemic immune response. Disrupting TWEAK/Fn14 interactions may be an innovative kidney-protective approach for the treatment of lupus nephritis and other antibody-induced renal diseases.

Elevated expression of Fn14 in non-small cell lung cancer correlates with activated EGFR and promotes tumor cell migration and invasion.

Lung cancer is the leading cause of cancer deaths worldwide; approximately 85% of these cancers are non-small cell lung cancer (NSCLC). Patients with NSCLC frequently have tumors harboring somatic mutations in the epidermal growth factor receptor (EGFR) gene that cause constitutive receptor activation. These patients have the best clinical response to EGFR tyrosine kinase inhibitors (TKIs). Herein, we show that fibroblast growth factor-inducible 14 (Fn14; TNFRSF12A) is frequently overexpressed in NSCLC tumors, and Fn14 levels correlate with p-EGFR expression. We also report that NSCLC cell lines that contain EGFR-activating mutations show high levels of Fn14 protein expression. EGFR TKI treatment of EGFR-mutant HCC827 cells decreased Fn14 protein levels, whereas EGF stimulation of EGFR wild-type A549 cells transiently increased Fn14 expression. Furthermore, Fn14 is highly expressed in EGFR-mutant H1975 cells that also contain an EGFR TKI-resistance mutation, and high TKI doses are necessary to reduce Fn14 levels. Constructs encoding EGFRs with activating mutations induced Fn14 expression when expressed in rat lung epithelial cells. We also report that short hairpin RNA-mediated Fn14 knockdown reduced NSCLC cell migration and invasion in vitro. Finally, Fn14 overexpression enhanced NSCLC cell migration and invasion in vitro and increased experimental lung metastases in vivo. Thus, Fn14 may be a novel therapeutic target for patients with NSCLC, in particular for those with EGFR-driven tumors who have either primary or acquired resistance to EGFR TKIs.

The cytokine tumor necrosis factor-like weak inducer of apoptosis and its receptor fibroblast growth factor-inducible 14 have a neuroprotective effect in the central nervous system.

BACKGROUND: Cerebral cortical neurons have a high vulnerability to the harmful effects of hypoxia. However, the brain has the ability to detect and accommodate to hypoxic conditions. This phenomenon, known as preconditioning, is a natural adaptive process highly preserved among species whereby exposure to sub-lethal hypoxia promotes the acquisition of tolerance to a subsequent lethal hypoxic injury. The cytokine tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor-inducible 14 (Fn14) are found in neurons and their expression is induced by exposure to sub-lethal hypoxia. Accordingly, in this work we tested the hypothesis that the interaction between TWEAK and Fn14 induces tolerance to lethal hypoxic and ischemic conditions. METHODS: Here we used in vitro and in vivo models of hypoxic and ischemic preconditioning, an animal model of transient middle cerebral artery occlusion and mice and neurons genetically deficient in TWEAK, Fn14, or tumor necrosis factor alpha (TNF-α) to investigate whether treatment with recombinant TWEAK or an increase in the expression of endogenous TWEAK renders neurons tolerant to lethal hypoxia. We used enzyme-linked immunosorbent assay to study the effect of TWEAK on the expression of neuronal TNF-α, Western blot analysis to investigate whether the effect of TWEAK was mediated by activation of mitogen-activated protein kinases and immunohistochemical techniques and quantitative real-time polymerase chain reaction analysis to study the effect of TWEAK on apoptotic cell death. RESULTS: We found that either treatment with recombinant TWEAK or an increase in the expression of TWEAK and Fn14 induce hypoxic and ischemic tolerance in vivo and in vitro. This protective effect is mediated by neuronal TNF-α and activation of the extracellular signal-regulated kinases 1 and 2 pathway via phosphorylation and inactivation of the B-cell lymphoma 2-associated death promoter protein. CONCLUSIONS: Our work indicate that the interaction between TWEAK and Fn14 triggers the activation of a cell signaling pathway that results in the induction of tolerance to lethal hypoxia and ischemia. These data indicate that TWEAK may be a potential therapeutic strategy to protect the brain from the devastating effects of an ischemic injury.

Resolving TRPV1- and TNF-α-mediated spinal cord synaptic plasticity and inflammatory pain with neuroprotectin D1.

Mechanisms of inflammatory pain are not fully understood. We investigated the role of TRPV1 (transient receptor potential subtype V1) and TNF-α, two critical mediators for inflammatory pain, in regulating spinal cord synaptic transmission. We found in mice lacking Trpv1 the frequency but not the amplitude of spontaneous EPSCs (sEPSCs) in lamina II neurons of spinal cord slices is reduced. Further, C-fiber-induced spinal long-term potentiation (LTP) in vivo is abolished in Trpv1 knock-out mice. TNF-α also increases sEPSC frequency but not amplitude in spinal outer lamina II (lamina IIo) neurons, and this increase is abolished in Trpv1 knock-out mice. Single-cell PCR analysis revealed that TNF-α-responding neurons in lamina IIo are exclusively excitatory (vGluT2(+)) neurons. Notably, neuroprotectin-1 (NPD1), an anti-inflammatory lipid mediator derived from ω-3 polyunsaturated fatty acid (docosahexaenoic acid), blocks TNF-α- and capsaicin-evoked sEPSC frequency increases but has no effect on basal synaptic transmission. Strikingly, NPD1 potently inhibits capsaicin-induced TRPV1 current (IC(50) = 0.4 nm) in dissociated dorsal root ganglion neurons, and this IC(50) is ≈ 500 times lower than that of AMG9810, a commonly used TRPV1 antagonist. NPD1 inhibition of TRPV1 is mediated by GPCRs, since the effects were blocked by pertussis toxin. In contrast, NPD1 had no effect on mustard oil-induced TRPA1 currents. Spinal injection of NPD1, at very low doses (0.1-10 ng), blocks spinal LTP and reduces TRPV1-dependent inflammatory pain, without affecting baseline pain. NPD1 also reduces TRPV1-independent but TNF-α-dependent pain hypersensitivity. Our findings demonstrate a novel role of NPD1 in regulating TRPV1/TNF-α-mediated spinal synaptic plasticity and identify NPD1 as a novel analgesic for treating inflammatory pain.

IL-17 induces hyperalgesia via TNF-dependent neutrophil infiltration.

Interleukin-17 (IL-17) and tumour necrosis factor-α (TNF) are critical in the pathogenesis of arthritis but their relationship during inflammatory pain has received limited attention. We aimed to establish whether IL-17 can induce hyperalgesia in acute conditions, and investigated the role of TNF in mediating the pain response. Hyperalgesia was elicited in C57BL/6 mice by injection of recombinant IL-17, TNF or vehicle into the plantar tissue. Elevated pain was measured by the Hargreaves test for thermal hyperalgesia and Linton incapacitance tester for weight-bearing change. Cellular infiltration during hyperalgesia was determined by histological analysis and myeloperoxidase assay. IL-17 was found to induce hyperalgesia, but this was dependent on neutrophil migration and TNF binding to TNF receptor 1 (TNFR1). Because TNF-induced hyperalgesia was also dependent on neutrophil migration, the relationship between the resident fibroblasts, the cytokines and the migrating neutrophils was further investigated. By means of an air pouch model of cell migration, it was established that IL-17-induced neutrophil infiltration was dependent of TNF/TNFR1 as this interaction was required for the induction of the chemokine keratinocyte chemoattractant. These findings suggest that IL-17 causes acute hyperalgesia indirectly by inducing TNF from resident cells. The subsequent production of keratinocyte chemoattractant then triggers neutrophil chemotaxis to the plantar tissue, releasing algesic mediators locally to sensitise the nerve.

Linear ubiquitination prevents inflammation and regulates immune signalling.

Members of the tumour necrosis factor (TNF) receptor superfamily have important functions in immunity and inflammation. Recently linear ubiquitin chains assembled by a complex containing HOIL-1 and HOIP (also known as RBCK1 and RNF31, respectively) were implicated in TNF signalling, yet their relevance in vivo remained uncertain. Here we identify SHARPIN as a third component of the linear ubiquitin chain assembly complex, recruited to the CD40 and TNF receptor signalling complexes together with its other constituents, HOIL-1 and HOIP. Mass spectrometry of TNF signalling complexes revealed RIP1 (also known as RIPK1) and NEMO (also known as IKKγ or IKBKG) to be linearly ubiquitinated. Mutation of the Sharpin gene (Sharpin(cpdm/cpdm)) causes chronic proliferative dermatitis (cpdm) characterized by inflammatory skin lesions and defective lymphoid organogenesis. Gene induction by TNF, CD40 ligand and interleukin-1ß was attenuated in cpdm-derived cells which were rendered sensitive to TNF-induced death. Importantly, Tnf gene deficiency prevented skin lesions in cpdm mice. We conclude that by enabling linear ubiquitination in the TNF receptor signalling complex, SHARPIN interferes with TNF-induced cell death and, thereby, prevents inflammation. Our results provide evidence for the relevance of linear ubiquitination in vivo in preventing inflammation and regulating immune signalling.

TNF is necessary for castration-induced prostate regression, whereas TRAIL and FasL are dispensable.

TNF, a proinflammatory and immune-regulatory cytokine, is a potent apoptotic stimulus in vitro. However, there have been few examples of a physiologic role for TNF-induced apoptosis in vivo. Here, we describe a novel role for TNF in prostate epithelial cell apoptosis after androgen withdrawal. Employing high-resolution serial magnetic resonance imaging to measure mouse prostate volume changes over time, we demonstrate that the extent of castration-induced prostate regression is significantly reduced in mice null for either the Tnf or Tnfr1 genes but not mice deficient for TNF-related apoptosis-inducing ligand or Fas signaling. Wild-type mice receiving soluble TNF (sTNF) receptor 2 (to bind TNF and block signaling) before castration exhibit an identical reduction of prostate regression. Together, these data indicate that uniquely among known extrinsic death signals, TNF is required for castration-induced prostate regression. Additionally, membrane-bound TNF protein and stromal cell specific TNF mRNA levels increase in rat prostate after castration. This is consistent with a paracrine role for TNF in prostate regression. When injected into the peritoneum of Tnf(-/-) mice at the time of castration, sTNF restores normal levels of prostate regression. However, wild-type mice receiving sTNF in the absence of castration do not exhibit prostate regression, indicating that TNF alone is not sufficient but acts in the context of additional castration-induced signals. These findings support a physiologic role for TNF in prostate regression after androgen withdrawal. Understanding this role may lead to novel therapies for prostate cancer.