Paul M. Radford

Mutant forms of tumour necrosis factor receptor I that occur in TNF‐receptor‐associated periodic syndrome retain signalling functions but show abnormal behaviour

Tumour necrosis factor (TNF)‐receptor‐associated periodic syndrome (TRAPS) is a hereditary autoinflammatory disorder involving autosomal‐dominant missense mutations in TNF receptor superfamily 1A (TNFRSF1A) ectodomains. To elucidate the molecular effects of TRAPS‐related mutations, we transfected HEK‐293 cells to produce lines stably expressing high levels of either wild‐type (WT) or single mutant recombinant forms of TNFRSF1A. Mutants with single amino acid substitutions in the first cysteine‐rich domain (CRD1) were produced both as full‐length receptor proteins and as truncated forms lacking the cytoplasmic signalling domain (Δsig). High‐level expression of either WT or mutant full‐length TNFRSF1A spontaneously induced apoptosis and interleukin‐8 production, indicating that the mutations in CRD1 did not abrogate signalling. Consistent with this, WT and mutant full‐length TNFRSF1A formed cytoplasmic aggregates that co‐localized with ubiquitin and chaperones, and with the signal transducer TRADD, but not with the inhibitor, silencer of death domain (SODD). Furthermore, as expected, WT and mutant Δsig forms of TNFRSF1A did not induce apoptosis or interleukin‐8 production. However, whereas the WT full‐length TNFRSF1A was expressed both in the cytoplasm and on the cell surface, the mutant receptors showed strong cytoplasmic expression but reduced cell‐surface expression. The WT and mutant Δsig forms of TNFRSF1A were all expressed at the cell surface, but a proportion of the mutant receptors were also retained in the cytoplasm and co‐localized with BiP. Furthermore, the mutant forms of surface‐expressed Δsig TNFRSF1A were defective in binding TNF‐α. We conclude that TRAPS‐related CRD1 mutants of TNFRSF1A possess signalling properties associated with the cytoplasmic death domain, but other behavioural features of the mutant receptors are abnormal, including intracellular trafficking and TNF binding.

Role of interleukin-6 in a patient with tumor necrosis factor receptor-associated periodic syndrome: assessment of outcomes following treatment with the anti-interleukin-6 receptor monoclonal antibody tocilizumab.

In this report, we describe treatment outcomes in the first case of a patient with tumor necrosis factor receptor-associated periodic syndrome (TRAPS) treated with the anti-interleukin-6 (anti-IL-6) receptor monoclonal antibody tocilizumab. Since IL-6 levels are elevated in TRAPS, we hypothesized that tocilizumab might be effective. The patient, a 52-year-old man with lifelong TRAPS in whom treatment with etanercept and anakinra had failed, was administered tocilizumab for 6 months, and the therapeutic response was assessed by measurement of monocyte CD16 expression and cytokine levels. Following treatment, the evolving acute attack was aborted and further attacks of TRAPS were prevented. The patient did not require corticosteroids and showed significant clinical improvement in scores for pain, stiffness, and well-being. Moreover, the acute-phase response diminished significantly with treatment. Monocyte CD16 expression was reduced and the numbers of circulating CD14+CD16+ and CD14++CD16- monocytes were transiently decreased. However, cytokine levels were not reduced. This case supports the notion of a prominent role for IL-6 in mediating the inflammatory attacks in TRAPS, but blockade of IL-6 did not affect the underlying pathogenesis. These preliminary findings require confirmation.

Islet glutamic acid decarboxylase modified by reactive oxygen species is recognized by antibodies from patients with type 1 diabetes mellitus

The generation of an autoimmune response against islet beta‐cells is central to the pathogenesis of type 1 diabetes mellitus, and this response is driven by the stimulation of autoreactive lymphocytes by components of the beta‐cells themselves. Reactive oxygen species (ROS) have been implicated in the beta‐cell destruction which leads to type 1 diabetes and may modify beta‐cell components so as to enhance their immunogenicity. We investigated the effects of oxidation reactions catalysed by copper or iron on the major beta‐cell autoantigen glutamic acid decarboxylase (GAD). Lysates of purified rat islets were exposed to copper or iron sulphate with or without hydrogen peroxide or ascorbic acid. Immunostaining showed that these treatments generated high molecular weight covalently linked aggregates containing GAD. These are not formed by intermolecular disulphide bonds between cysteine residues since they cannot be resolved into monomeric form when electrophoresed under extreme reducing conditions. There was no modification of insulin or pro‐insulin by ROS. The same oxidative changes to GAD could be induced in viable islet cells treated with copper sulphate and hydrogen peroxide, and thus the modifications are not an artefact of the catalysed oxidation of cell‐free lysates. Sera from patients with type 1 diabetes and stiffman syndrome containing GAD antibodies reacted predominantly with the highest molecular weight modified protein band of GAD: normal human sera did not precipitate GAD. Thus, oxidatively modified aggregates of GAD react with serum antibodies of type 1 diabetes patients and some SMS patients: this is consistent with oxidative modifications of autoantigens being relevant to the pathogenesis of type 1 diabetes.

Distinct antigenic features of linear epitopes at the N-terminus and C-terminus of 65 kDa glutamic acid decarboxylase (GAD65): implications for autoantigen modification during pathogenesis

Autoantibodies to 65 kDa glutamic acid decarboxylase (GAD65) are produced in many patients with autoimmune polyendocrine syndrome type II (APS‐II) or stiff‐man syndrome (SMS) and are heterogeneous in their epitope specificities, recognizing both conformational and linear determinants. Major linear epitopes of GAD, which are recognized by autoantibodies in a minority of these patients, occur in the N‐terminal and C‐terminal regions. We have investigated antibody recognition of the N‐ and C‐termini of GAD65 in relation to their structural features as an approach to understanding what modifications to the native GAD structure may occur that facilitate the generation of antibodies specific to linear epitopes in these regions during the autoimmune pathogenesis. A monoclonal antibody specific to the N‐terminus of GAD65 bound both native and denatured GAD in ELISA, whereas monoclonal and polyclonal antibodies specific to the C‐terminus of GAD bound only denatured GAD. These antibodies were epitope mapped using random peptide phage‐display libraries and the epitopes related to a previously proposed structural model of GAD65. This has led us to propose that the α‐helical secondary structure of the C‐terminus of GAD65 must be denatured to generate linear epitopes. In contrast, the N‐terminus is both surface exposed and linear in the native structure, but may be masked by membrane interactions, which must be broken to facilitate recognition by B cells.

Novel markers of inflammation identified in tumor necrosis factor receptor–associated periodic syndrome (TRAPS) by transcriptomic analysis of effects of TRAPS‐associated tumor necrosis factor receptor type I mutations in an endothelial cell line

OBJECTIVE To analyze the effects of tumor necrosis factor receptor-associated periodic syndrome (TRAPS)-associated mutant tumor necrosis factor receptor type I (TNFRI) expression in a cell type directly relevant to the inflammation in TRAPS, and to identify novel markers associated with mutant TNFRI expression. METHODS Transcriptome analysis on 30,000 human genes was performed on SK-Hep-1 human endothelial cells transfected with either wild-type (WT) or TRAPS-associated mutant TNFRI. Quantitative reverse transcriptase-polymerase chain reaction and protein expression levels measured by enzyme-linked immunosorbent assay verified transcriptional changes for selected genes both in supernatants from cells expressing mutant TNFRI and in patient plasma. RESULTS Cells expressing mutant TNFRI showed up-regulation of multiple proinflammatory genes relative to WT transfectants, including genes for pentraxin 3, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, CCL2, and CCL5, which were also expressed as proteins. In addition, the expression of most of these markers was increased in the plasma and peripheral blood mononuclear cells from TRAPS patients relative to those from healthy controls. The cysteine mutations (C33Y and C52F), which are associated with a more severe clinical phenotype, induced more genes than the low-penetrance mutation R92Q, which is associated with a milder phenotype. The expression of most genes was induced by a death domain (DD)-dependent mechanism, since they were not induced by expression of TNFRI mutants with an inactivated DD. CONCLUSION TRAPS-associated TNFRI mutants induce the expression of multiple genes encoding inflammatory molecules, cellular receptors, transcription factors, and regulators of apoptosis in endothelial cells that require the cytoplasmic signaling properties of the receptor. Different mutants have specific expression profiles, indicating mutation-specific effects. The expression of some of these markers was also elevated in samples from TRAPS patients.

Tumour necrosis factor receptor I blockade shows that TNF-dependent and TNF-independent mechanisms synergise in TNF receptor associated periodic syndrome

TNF receptor associated periodic syndrome (TRAPS) is an autoinflammatory disease involving recurrent episodes of fever and inflammation. It is associated with autosomal dominant mutations in TNF receptor superfamily 1A gene localised to exons encoding the ectodomain of the p55 TNF receptor, TNF receptor‐1 (TNFR1). The aim of this study was to investigate the role of cell surface TNFR1 in TRAPS, and the contribution of TNF‐dependent and TNF‐independent mechanisms to the production of cytokines. HEK‐293 and SK‐HEP‐1 cell lines were stably transfected with WT or TRAPS‐associated variants of human TNF receptor superfamily 1A gene. An anti‐TNFR1 single domain antibody (dAb), and an anti‐TNFR1 mAb, bound to cell surface WT and variant TNFR1s. In HEK‐293 cells transfected with death domain‐inactivated (R347A) TNFR1, and in SK‐HEP‐1 cells transfected with normal (full‐length) TNFR1, cytokine production stimulated in the absence of exogenous TNF by the presence of certain TNFR1 variants was not inhibited by the anti‐TNFR1 dAb. In SK‐Hep‐1 cells, specific TRAPS mutations increased the level of cytokine response to TNF, compared to WT, and this augmented cytokine production was suppressed by the anti‐TNFR1 dAb. Thus, TRAPS‐associated variants of TNFR1 enhance cytokine production by a TNF‐independent mechanism and by sensitising cells to a TNF‐dependent stimulation. The TNF‐dependent mechanism requires cell surface expression of TNFR1, as this is blocked by TNFR1‐specific dAb.

Measuring and modelling the response of Klebsiella pneumoniae KPC prey to Bdellovibrio bacteriovorus predation, in human serum and defined buffer

In worldwide conditions of increasingly antibiotic-resistant hospital infections, it is important to research alternative therapies. Bdellovibrio bacteriovorus bacteria naturally prey on Gram-negative pathogens, including antibiotic-resistant strains and so B. bacteriovorus have been proposed as “living antibiotics” to combat antimicrobially-resistant pathogens. Predator-prey interactions are complex and can be altered by environmental components. To be effective B. bacteriovorus predation needs to work in human body fluids such as serum where predation dynamics may differ to that studied in laboratory media. Here we combine mathematical modelling and lab experimentation to investigate the predation of an important carbapenem-resistant human pathogen, Klebsiella pneumoniae, by B. bacteriovorus in human serum versus buffer. We show experimentally that B. bacteriovorus is able to reduce prey numbers in each environment, on different timescales. Our mathematical model captures the underlying dynamics of the experimentation, including an initial predation-delay at the predator-prey-serum interface. Our research shows differences between predation in buffer and serum and highlights both the potential and limitations of B. bacteriovorus acting therapeutically against K. pneumoniae in serum, informing future research into the medicinal behaviours and dosing of this living antibacterial.

Functional consequences of disease-associated mutations in TNFR1 elucidated by transcriptome analysis.

Mutations in the TNFRSF1A (TNF receptor 1) gene cause the autosomal dominant, autoinflammatory tumour necrosis factor receptor-associated periodic syndrome (TRAPS). TRAPS is characterised by recurring attacks of fever, abdominal pain, arthralgia, myalgia, conjunctivitis, migratory skin lesions and systemic amyloidosis. The exact mechanism by which this periodic fever is triggered has yet to be elucidated. Over the last decade, several studies with patient and transfected cells have attempted to dissect the molecular mechanisms induced by mutant receptor expression and resulted in several hypotheses being proposed to explain the disease pathogenesis. Our recent data suggest that cellular expression of mutant TNFR1 per se is a signal for inflammatory changes. We hypothesised that different mutations can lead to the activation of distinct intracellular signalling pathways with a pro-inflammatory outcome in a TNF-independent fashion. To test this hypothesis, we investigated how different TNFR1 mutants affect global gene expression in epithelial and endothelial cell models. Compared to WT, cells expressing mutant TNFR1 showed downregulation of genes that suppress immune responses and upregulation of multiple genes involved in mediating inflammation. Both cell type-specific and mutant-specific profiles were identified. We have identified a number of novel genes that may play a role in the pathogenesis of TRAPS. Further studies with patient cells and in vivo models are needed to clarify relevance for disease pathogenesis and assist in the identification of new drug targets.

A signalome screening approach in the autoinflammatory disease TNF receptor associated periodic syndrome (TRAPS) highlights the anti-inflammatory properties of drugs for repurposing

&NA; TNF receptor associated periodic syndrome (TRAPS) is an autoinflammatory disease caused by mutations in TNF Receptor 1 (TNFR1). Current therapies for TRAPS are limited and do not target the pro‐inflammatory signalling pathways that are central to the disease mechanism. Our aim was to identify drugs for repurposing as anti‐inflammatories based on their ability to down‐regulate molecules associated with inflammatory signalling pathways that are activated in TRAPS. This was achieved using rigorously optimized, high through‐put cell culture and reverse phase protein microarray systems to screen compounds for their effects on the TRAPS‐associated inflammatory signalome. 1360 approved, publically available, pharmacologically active substances were investigated for their effects on 40 signalling molecules associated with pro‐inflammatory signalling pathways that are constitutively upregulated in TRAPS. The drugs were screened at four 10‐fold concentrations on cell lines expressing both wild‐type (WT) TNFR1 and TRAPS‐associated C33Y mutant TNFR1, or WT TNFR1 alone; signalling molecule levels were then determined in cell lysates by the reverse‐phase protein microarray. A novel mathematical methodology was developed to rank the compounds for their ability to reduce the expression of signalling molecules in the C33Y‐TNFR1 transfectants towards the level seen in the WT‐TNFR1 transfectants. Seven high‐ranking drugs were selected and tested by RPPA for effects on the same 40 signalling molecules in lysates of peripheral blood mononuclear cells (PBMCs) from C33Y‐TRAPS patients compared to PBMCs from normal controls. The fluoroquinolone antibiotic lomefloxacin, as well as others from this class of compounds, showed the most significant effects on multiple pro‐inflammatory signalling pathways that are constitutively activated in TRAPS; lomefloxacin dose‐dependently significantly reduced expression of 7/40 signalling molecules across the Jak/Stat, MAPK, NF‐&kgr;B and PI3K/AKT pathways. This study demonstrates the power of signalome screening for identifying candidates for drug repurposing. Graphical abstract Figure. No caption available.