David J. MacEwan

Experimental design and analysis and their reporting: new guidance for publication in BJP

This Editorial is part of a series. To view the other Editorials in this series, visit: http://onlinelibrary.wiley.com/doi/10.1111/bph.12956/abstract; http://onlinelibrary.wiley.com/doi/10.1111/bph.12954/abstract; http://onlinelibrary.wiley.com/doi/10.1111/bph.12955/abstract and http://onlinelibrary.wiley.com/doi/10.1111/bph.13112/abstract

TNF receptor subtype signalling: Differences and cellular consequences

Tumour necrosis factor-alpha (TNF alpha) is a multifunctional cytokine belonging to a family of ligands with an associated family of receptor proteins. The pleiotropic actions of TNF range from proliferative responses such as cell growth and differentiation, to inflammatory effects and the mediation of immune responses, to destructive cellular outcomes such as apoptotic and necrotic cell death mechanisms. Activated TNF receptors mediate the association of distinct adaptor proteins that regulate a variety of signalling processes including kinase or phosphatase activation, lipase stimulation, and protease induction. Moreover, the cytokine regulates the activities of transcription factors, heterotrimeric or monomeric G-proteins and calcium ion homeostasis in order to orchestrate its cellular functions. This review addresses the structural basis of TNF signalling, the pathways employed with their cellular consequences, and focuses on the specific role played by each of the two TNF receptor isotypes, TNFR1 and TNFR2.

TNF ligands and receptors – a matter of life and death

It had been known for some time that tumour masses which had become contaminated by a bacterial infection would on occasion regress and disappear. It was thought that the bacteria were releasing a factor which would make the tumour necrotic and whither. This factor was termed tumour necrosis factor (TNF) (Old, 1985). It was not until more recent advances in immunology that it became clear that antigens from the bacterial invader (notably lipopolysaccharide LPS) were causing the release of the patients own TNF which could cause the tumour regression. The hunt was on to isolate this TNF which could be used as a magic therapy to control cancer cell growth and persistence, plus enhance the academic understanding of the ways by which a cell could die. It was discovered that TNF and lymphotoxin (LT) were products from macrophages and lymphocytes that were capable of lysing many cell types including some tumour cells (Carswell et al., 1975; Granger et al., 1969). TNF was also found to be identical to the protein cachectin, which was known to be involved in the fever and muscle wastage seen in cancer patients (Beutler et al., 1985). Hence, the role TNF played in a range of physiological actions was important and the hunt was on to identify TNF and related molecules such as LT. Modern techniques have since allowed the isolation, characterization and cloning of the genes for TNF which is structurally related to LT plus an expanding family of TNF-like ligands (Table 1). These cytokine molecules include ligands such as Fas, CD40 and RANK which cause wide-ranging long-term cellular activities in cells such as differentiation, proliferation or death. Evolution has created this TNF superfamily of cytokines to control and manipulate the immune system, modulating processes such as haematopoiesis, antibody production, or short- and long-term immunity. It is only through its quirky tumour-killing characteristic that TNF cytokine was first identified and may still hold the key to effective tumour therapy. As the majority of information has been gained about TNF and it is the archetypal cytokine of the superfamily, displaying the greatest range of cellular actions, this review will focus on the molecular aspects and biological role of TNF signalling. Table 1 TNF ligand superfamily

Actions of cannabinoid receptor ligands on rat cultured sensory neurones: implications for antinociception

Cannabinoids modulate nociceptive processing in models of acute, inflammatory and neuropathic pain. We have investigated the location and function of cannabinoid receptors on cultured neonatal dorsal root ganglion (DRG) neurones and F-11 cells, a dorsal root ganglionxneuroblastoma hybridoma which displays several of the features of authentic DRG neurones. CB(1) receptor immunolabelling was observed on the cell bodies and as fine puncta on processes of both cultured DRG neurones and F-11 cells. Additionally, fluorescence-activated cell sorting (FACS) analysis provided evidence that both CB(1) and CB(2) receptors are expressed on populations of cells within the cultured DRG and F-11 cells. The cannabinoid receptor agonist (+)-WIN55212 (10 and 100 nM) inhibited the mean voltage-activated Ca(2+) current in DRG neurones by 21% and 30%, respectively. The isomer, (-)-WIN55212 (10 and 100 nM) produced significantly less inhibition of 6% and 10% respectively. The CB(1) selective receptor antagonist SR141716A (100 nM) enhanced the peak high voltage-activated Ca(2+) current by 24% and simultaneous application of SR141716A (100 nM) and (+)-WIN55212 (100 nM) resulted in a significant attenuation of the inhibition obtained with (+)-WIN55212 alone. These data give functional evidence for the hypothesis that the analgesic actions of cannabinoids may be mediated by presynaptic inhibition of transmitter release in sensory neurones.

TNF-α receptors simultaneously activate Ca2+ mobilisation and stress kinases in cultured sensory neurones

The cytokine tumour necrosis factor-alpha (TNF) has been implicated in autoimmune diseases and may play an indirect role in activation of pain pathways. In this study we have investigated the possibility that TNF directly activates cultured neonatal rat dorsal root ganglion (DRG) neurones and provides a signalling pathway from cells in the immune system such as macrophages to sensory neurones. Expression of TNF receptor subtypes (TNFR1 and TNFR2) on sensory neurones was identified using immunohistochemistry, fluorescence-activated cell sorting analysis and RT-PCR. Biochemical and immunocytochemical analysis showed that TNF activated p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK) but not p42/p44 MAPK. TNF treatment evoked transient Ca2+-dependent inward currents in 70% of DRG neurones. These TNF-evoked currents were significantly attenuated by ryanodine or thapsigargin or by inclusion of BAPTA in the patch pipette solution. Responses were also evoked in subpopulations of cultured DRG neurones by human mutant TNFs that cross-reacted with rat receptors and selectively activated TNFR1 or TNFR2 subtypes. TNF-evoked transient increases in [Ca2+]i were also detected in 34% of fura-2-loaded DRG neurones. The link between TNF receptor activation and Ca2+ release from stores remains to be elucidated. However, responses to TNF were mimicked by sphingolipids, including sphingosine-1-phosphate, which evoked a transient rises in [Ca2+]i in a pertussis toxin-insensitive manner in fura-2-loaded DRG neurones. We conclude that distinct receptors TNFR1 and TNFR2 are expressed on cultured DRG neurones and that they are functionally linked to intracellular Ca2+ mobilisation, a response that may involve sphingolipid signalling.

Lipopolysaccharide-Induced Expression of NAD(P)H:Quinone Oxidoreductase 1 and Heme Oxygenase-1 Protects against Excessive Inflammatory Responses in Human Monocytes

Monocytes play a central role in the immunopathological effects of sepsis. This role is mediated by production of the cytokines TNF-α and IL-1β. The transcription factor NF-E2-related factor 2 (Nrf2) regulates innate immune responses in various experimental disease models. Presently, the role of Nrf2-regulated genes in LPS-treated human monocytes is not well defined. Herein we show that Nrf2 mediates a significant regulation of LPS-induced inflammatory responses. Analysis of Nrf2-regulated gene expression in human monocytes showed that LPS induced the expression of the phase II detoxification gene NAD(P)H:quinone oxidoreductase 1 (NQO1). Furthermore, NQO1 mRNA or protein expression in response to LPS was regulated by Nrf2. Silencing Nrf2 expression in human monocytes inhibited LPS-induced NQO1 expression; however, in contrast, it significantly increased TNF and IL-1β production. Silencing expression of NQO1 alone, or in combination with heme oxygenase-1 (HO-1) silencing, markedly increased LPS-induced TNF and IL-1β expression. Additionally, overexpression of NQO1 and/or HO-1 inhibited LPS-induced TNF and IL-1β expression. These results show for the first time that LPS induces NQO1 and HO-1 expression in human monocytes via Nrf2 to modulate their inflammatory responsiveness, thus providing novel potential therapeutic strategies for the treatment of sepsis.

HO-1 underlies resistance of AML cells to TNF-induced apoptosis

In human monocytes, tumor necrosis factor (TNF) induces a proinflammatory response. In NF-kappaB-inhibited monocytes, TNF stimulates cell death/apoptosis. In the present study, we analyzed the response of acute myeloid leukemia (AML) cells to TNF stimulation in conjunction with NF-kappaB inhibition. In all AML-derived cells tested, NF-kappaB-inhibited cells were resistant to TNF-induced apoptosis. Further investigation revealed that the cytoprotective gene heme oxygenase-1 (HO-1) was induced in NF-kappaB-inhibited AML cells in response to TNF stimulation, and HO-1 was responsible for the resistance of AML cells to the cytotoxic actions of TNF. Moreover, after transfection with HO-1 siRNA, the resistance to TNF-induced cell death signals of AML cells was removed. The HO-1 promoter region contains antioxidant-response elements that can bind the transcription factor NF-E2-related factor 2 (Nrf2). We further demonstrated that Nrf2 was activated by TNF under NF-kappaB-inhibited conditions, to play the major role in up-regulating HO-1 expression and ultimately the fate of AML cells. These results demonstrate a novel mechanism by which TNF-induced cell death is inhibited in AML cells through the induction of HO-1, via Nrf2 activation.

The high Nrf2 expression in human acute myeloid leukemia is driven by NF-κB and underlies its chemo-resistance

NF-E2-related factor 2 (Nrf2) transcription factor regulates a range of cytoprotective transcriptional responses, preventing further cellular injury by removing biochemical damage and renewing tissue. Here we show that acute myeloid leukemia (AML) cells possess greater constitutive nuclear levels of Nrf2 than normal control CD34(+) cells because of an imbalance between mRNA expression levels of Nrf2 and its inhibitor Keap1 but not through their somatic mutation. Elevated Nrf2 was reduced by NF-κB inhibitors. Using promoter assays, ChIP and siRNA knockdown, we demonstrated NF-κB subunits p50 and p65 induce transcription of Nrf2 in AML cells at a specific promoter κB-site and that long-term lentiviral miRNA-knockdown of Nrf2 significantly reduced clonogenicity of AML patient cells and improved their chemotherapeutic responsiveness. Normal physiologic Nrf2 protects cells from damage, but here we have exposed aberrant continuous nuclear activation of Nrf2 in AML that allows cell survival, even against cytotoxic chemotherapeutics. We show for the first time that Nrf2, an important regulator of several biologic processes involved in the progression of cancer, has abnormal NF-κB-driven constitutive expression in AML. Such a mechanism allows for a greater cytoprotective response in human AML cells and encourages their evasion of chemotherapy-induced cytotoxicity, which is necessary for improved clinical outcomes.

BTK inhibitor ibrutinib is cytotoxic to myeloma and potently enhances bortezomib and lenalidomide activities through NF-κB

Ibrutinib (previously known as PCI-32765) has recently shown encouraging clinical activity in chronic lymphocytic leukaemia (CLL) effecting cell death through inhibition of Brutons tyrosine kinase (BTK). In this study we report for the first time that ibrutinib is cytotoxic to malignant plasma cells from patients with multiple myeloma (MM) and furthermore that treatment with ibrutinib significantly augments the cytotoxic activity of bortezomib and lenalidomide chemotherapies. We describe that the cytotoxicity of ibrutinib in MM is mediated via an inhibitory effect on the nuclear factor-κB (NF-κB) pathway. Specifically, ibrutinib blocks the phosphorylation of serine-536 of the p65 subunit of NF-κB, preventing its nuclear translocation, resulting in down-regulation of anti-apoptotic proteins Bcl-xL, FLIP(L) and survivin and culminating in caspase-mediated apoptosis within the malignant plasma cells. Taken together these data provide a platform for clinical trials of ibrutinib in myeloma and a rationale for its use in combination therapy, particularly with bortezomib.

Differential activation of nuclear factor-κB by tumour necrosis factor receptor subtypes. TNFR1 predominates whereas TNFR2 activates transcription poorly

Tumour necrosis factor‐α (TNF‐α) signals though two receptors, TNFR1 and TNFR2. TNFR1 has a role in cytotoxicity, whereas TNFR2 regulates death responses or proliferation. TNF activates pro‐inflammatory transcription factor nuclear factor‐κB (NF‐κB) by uncertain signalling mechanisms. Here we report the contribution of each TNFR towards the NF‐κB activation processes. In human cells expressing endogenous or exogenous TNFR2, in addition to TNFR1, we found both TNFRs capable of activating NF‐κB, as measured by IκBα (inhibitor of NF‐κB) degradation, electrophoretic mobility shift assay and NF‐κB gene reporter assays. TNFR2 activation did not degrade IκBβ. However, TNF‐effects on NF‐κB activation occurred predominantly through TNFR1, with TNFR2 activating the transcription factor poorly.