Tumor necrosis factor receptor–associated periodic syndrome (TRAPS; previously known as familial Hibernian fever) is an autosomal-dominant autoinflammatory condition caused by mutations in the TNFRSF1A gene (1). Autoinflammatory disorders are defined by spontaneously relapsing and remitting bouts of systemic inflammation in the absence of pathogens, autoantibodies, or antigen-specific T cells. Examples include both monogenic disorders (e.g., TRAPS, familial Mediterranean fever, Muckle-Wells syndrome, and hyperimmunoglobulinemia D with periodic fever syndrome [HIDS]) and multifactorial disorders (e.g., Crohn's disease and Behçet's disease) associated with mutations of genes involved in inflammation and/or apoptosis. TRAPS is characterized by recurrent periodic fevers, abdominal pain, skin lesions, conjunctivitis, and myalgia; AA amyloidosis develops in some patients, leading to renal problems.
Thus far, more than 50 different TNFRSF1A mutations have been described that are associated with TRAPS (INFEVERS database; online at http://fmf.igh.cnrs.fr/infevers) (2, 3), with the majority (>90%) being single-nucleotide missense mutations within exons 2, 3, 4, and 6. Most mutations are located within the extracellular region in cysteine-rich domains, with I170N being an exception, because it is located very close to the receptor cleavage site (4). Although the majority of mutations are fully penetrant, there are also some low-penetrance variants (e.g., P46L and R92Q) in which certain family members are symptomatic whereas others may remain unaffected despite carrying the same variant (5). These low-penetrance TNFRSF1A variants are also observed in the background population (6), indicating a possible role in inflammation in general.
The cellular mechanisms by which TNFRSF1A mutations lead to clinical disease are still largely unknown. NF-κB is a ubiquitous transcription factor activated by proinflammatory stimuli (7), which in turn leads to the up-regulation of a large number of genes involved in inflammation and apoptosis, including TNFα (Figure 1). Not only does activation of NF-κB stimulate the production of TNFα, but there is a positive feedback mechanism whereby binding of TNFα to its cell surface receptor augments the activity of NF-κB (8–10). NF-κB, however, is not a single molecule but consists of complexes of different combinations of the subunits RelA (p65), RelB, c-Rel, p50, and p52, each with distinct DNA-binding characteristics (11).
Figure 1. Cell surface signaling from tumor necrosis factor receptor I (TNFRI) leads to NF-κB activation and inflammatory response. Binding of TNFα to the trimeric form of TNFRI results in conformational changes in the intracellular region of the receptor, leading to rapid recruitment of several adaptor proteins including FADD and receptor-interacting protein (RIP). This initiates signaling cascades that lead to either caspase-mediated apoptosis or NF-κB activation and inflammatory response. NF-κB activation is characterized by dissociation from IκBα, following IKK complex–mediated phosphorylation and NF-κB–essential modulator (NEMO)–dependent ubiquitin (Ub) ligase–mediated ubiquitination, which leads to proteasomal degradation. The p65/p50 heterodimer subunits then translocate to the nucleus, where they bind to DNA target sequences of a number of cytokines and additional factors that are associated with inflammation. TRAPS-associated mutations are primarily found in the extracellular cysteine-rich domains (CRD) of TNFRI.
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TNFRI cell-signaling studies have, to date, been difficult to assess, due in part to proapoptotic consequences from overexpression studies. In addition, researchers have resorted to the use of constructs with cytoplasmic truncations of TNFRI that might have a major impact on protein structure, folding, and conformation. In this study, we set out to examine NF-κB activity in C73R and R92Q TNFRI mutant cells from patients with TRAPS, and also in a P46L TNFRI mutant cell line from a patient with the HIDS form of periodic fever with additional in vitro and in vivo evidence of TNFRI dysfunction, in order to determine whether signaling abnormalities could explain the variability in disease penetrance.
After finding increased NF-κB activity in peripheral blood mononuclear cells (PBMCs) from patients with TRAPS, we then investigated whether this might be attributable to increased cell surface expression and activity of TNFRI. Until now, reports have been contradictory as to whether TRAPS mutations lead to increased or decreased TNFRI expression. Studies reliant on transfection of complementary DNA vectors into immortalized cell lines demonstrated restricted cell surface expression of mutant TNFRI (12, 13). Similarly, the use of transfected mutant TNFRI–containing plasmids has been reported to result in abnormal disulfide-linked TNFR oligomerization, retention in the endoplasmic reticulum, and diminished NF-κB signaling (14). We used primary cells from patients with TRAPS mutations to answer this question more directly.
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- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
Early reports suggested that a TNFRI shedding defect was one of the principal molecular mechanisms resulting in TRAPS (1). However, it is still unclear whether impaired cytokine receptor clearance is the pathophysiologic basis for a majority or just a subset of TRAPS-associated mutations. Several recent studies using transfected cells suggested other possible mechanisms of the disease: reduced cell-surface expression of mutant receptors (13), an abnormal oligomerization with ER retention (14), decreased binding of mutant forms of receptor to TNFα (12), ligand-independent signaling (12, 16), and a reduction in TNF-induced apoptosis (23, 24). Although some of the findings were consistent among different groups, others were variable, most probably due to various experimental conditions (e.g., full-length versus truncated mutant constructs, different cell types used for in vitro transfections). Moreover, few studies used mutant cells from patients for ex vivo experiments, and there also has been some disagreement regarding apoptosis and shedding defects that may be explained by fibroblast versus PBMC cell type effects (25).
In addition to the above, one of the major unresolved issues is whether the NF-κB pathway is up-regulated or down-regulated in the presence of TRAPS-associated mutations. The NF-κB transcription factor consists of functional homodimers or heterodimers composed of its 5 monomeric family member subunits: RelA (p65), RelB, c-Rel, p50, and p52. TNFα is a widely studied inducer of NF-κB activity, with active NF-κB in turn modulating the expression of a range of genes associated with inflammation and immune responses (Figure 1). TNFα activity is mediated through 2 receptors, TNFRI and TNFRII, with mutations in the extracellular domains of the former being associated with the chronic inflammatory disorder, TRAPS. In the current study, we set out to examine NF-κB activity in PBMCs from patients with 3 TNFRSF1A mutations (C73R, P46L, and R92Q), in order to determine whether either TNFRI subcellular localization or intracellular signaling abnormalities could explain the variability in disease penetrance associated with these mutations.
Increased expression of the p50 subunit has previously been shown to play a role in the development of tolerance to lymphotoxin (26). Indeed, in lipopolysaccharide-tolerant monocytes, it has been shown that there is a 3-fold increase in p50 mobilization to the nucleus but no change in p65 status (27). Although both p50 and p65 bind DNA through a shared Rel homology domain, p65 also contains C-terminal transcription activator domains that are absent in p50. This has led to the suggestion that p50/p50 homodimers act as transcription repressors (28). Our data here show that in R92Q cells, basal levels of p65 were modestly elevated, which might in part help explain why this mutation can sometimes lead to disease association; nonetheless, basal levels of the p50 subunit were far higher. Moreover, in R92Q cells, the level of p65 is only very modestly elevated in response to TNFα stimulation relative to control cells. This likely reflects the presence of large quantities of p50/p50 homodimers bound to DNA of R92Q cells, thereby preventing significant high-penetrance immune activation.
It should be noted, however, that in addition to the aforementioned repressor action of p50, there are additional nuclear functions of p50. The IκB family member Bcl-3 performs a role similar to that of other IκB family members in removing NF-κB subunits from DNA. However, Bcl-3 also has the ability to form activating complexes with p50 on DNA (29, 30). Therefore, although p50 lacks a transcription activator domain, it might nevertheless still act as a transcription activator through Bcl-3 complex interactions. Indeed, p50 has been shown not only to interact with Bcl-3, but through this complex it can also interact with several additional transcription factors (31). Thus, the consequence of such abundant p50 localization to the nucleus in R92Q cells might be serving as a general proinflammatory activator through a number of off-target interactions, as well as acting to repress p65 NF-κB subunit signaling. Moreover, this might perhaps explain why there are low-penetrance associations between R92Q and several inflammatory diseases.
In contrast to R92Q cells, the C73R mutation in TNFRI results in significant translocation of the active p65 subunit of NF-κB. Furthermore, these data are highly reproducible, being pooled from 8 independent experiments using freshly isolated PBMCs from a mother and her daughter who possess this mutation. As is the case with C52R (32), fluorescence-activated cell sorting analysis of monocyte cell surface following proteinase activation has previously shown that there is no shedding defect in C73R cells (33). The results presented here show that cell surface localization can be influenced by mutations within the cysteine-rich domain (CRD), CRD2. This raises the possibility that there is a Golgi retention motif within this region, or in an area of the molecule that is influenced by mutations in this region, and that the C73R mutation abolishes this retention signal. Further study of other fully penetrant TRAPS mutations is, however, needed before we can accurately determine whether this is a common phenomenon of TRAPS mutations or is instead a specific characteristic of C73R.
The resulting effect of the C73R mutation is initiation of the NF-κB pathway, and this therefore links TNFRI cell surface expression to inflammation status. Interestingly, the P46L mutation would be predicted to alter bending in the protein secondary structure, which likely introduces a conformational change in loop 3 of the highly conserved CRD1 (6, 34). We suggest that although P46L cells contain high levels of TNFRI at the cell surface, they fail to show an enhanced response to inflammatory stimulation above control due to inactive receptor. We cannot, however, rule out the possibility that the mutation of mevalonate kinase in this patient contributed to the current findings, and thus the results should be interpreted with some caution. Nonetheless, in a separate patient with only the P46L mutation, it was previously demonstrated that P46L results in a TNFRI shedding defect (6) that is consistent with the current observations. Further study is required to determine precisely how TRAPS-associated conformational changes in TNFRI impact receptor assembly and trimerization.
In conclusion, this study is the first to show that the R92Q mutation associated with low-penetrance TRAPS likely results from p50/p50 NF-κB homodimer repression. The P46L mutation, however, does not alter p50 signaling but instead renders TNFRI nonfunctional. Because these observations were made on cells from patients with TRAPS and either the R92Q or P46L mutation, it would be of considerable interest for future studies to investigate TNFRI functionality and NF-κB signaling in asymptomatic carriers of these mutations. In contrast, the fully penetrant C73R mutation results in persistent elevated localization of functional TNFRI at the cell surface and is associated with increased TNFα induction of the proinflammatory intracellular NF-κB pathway. Thus, variation in NF-κB activity in PBMCs from patients with different TNFRI genotypes provides an explanation for the observed variation in clinical phenotype.
- Top of page
- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
Dr. Turner had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study design. Nedjai, Hitman, Yousaf, Chernajovsky, McDermott, Turner.
Acquisition of data. Nedjai, Stjernberg-Salmela, Pettersson, Ranki, Hawkins, Arkwright, McDermott, Turner.
Analysis and interpretation of data. Nedjai, Hitman, Yousaf, Chernajovsky, McDermott, Turner.
Manuscript preparation. Nedjai, Hitman, Yousaf, Chernajovsky, Stjernberg-Salmela, Pettersson, Ranki, Hawkins, Arkwright, McDermott, Turner.
Statistical analysis. Nedjai, Turner.