Background During the last several years we have studied five different dominantly-inherited autoinflammatory disorders. The first of these illnesses is the TNF receptor-associated periodic syndrome (TRAPS), which is characterized by prolonged attacks of fever, serositis, migratory rash and myalgia, arthritis, periorbital edema, conjunctivitis, and, in some patients, systemic amyloidosis. In 1999 we identified the first mutations in the gene (TNFRSF1A) encoding the 55 kDa tumor necrosis factor receptor (TNFR1) in families with dominantly-inherited recurrent fevers, and proposed the name TRAPS for this clinical condition. Initial mechanistic studies indicated a defect in the activation-induced shedding of the p55 (but not p75) TNF receptor, possibly leading to impaired homeostasis. Findings we published in the last year suggest an important additional mechanism of inflammation in these patients (see below). In 2002 we discovered dominantly-inherited de novo mutations in a second gene, initially named CIAS1 but now officially denoted NLRP3, in about 50% of patients with a disorder known as neonatal onset multisystem inflammatory disease (NOMID) or chronic infantile neurologic cutaneous and articular (CINCA) syndrome. Manifestations of NOMID/CINCA may include daily fevers, an urticaria-like skin rash, chronic aseptic meningitis, uveitis, papilledema, sensorineural hearing loss, mental retardation, patellar and epiphyseal long bone overgrowth, and systemic amyloidosis. Two milder conditions, familial cold autoinflammatory syndrome (FCAS) and Muckle-Wells syndrome (MWS), are caused by mutations in the same gene. NLRP3 encodes a protein, cryopyrin, that participates in a macromolecular complex called the inflammasome to regulate the activation of interleukin-1 (IL-1) beta. Collectively, all three diseases are known as the cryopyrin-associated periodic syndromes (CAPS). CAPS patients respond dramatically to inhibitors of IL-1 beta. A fifth dominantly-inherited autoinflammatory disorder, denoted the syndrome of pyogenic arthritis with pyoderma gangrenosum and acne (PAPA), is caused by mutations in a protein known as proline serine threonine phosphatase interacting protein (PSTPIP1). PAPA is characterized by episodes of sterile pyogenic arthritis, which can be destructive if not treated, formation of open, purulent ulcers of the skin (pyoderma gangrenosum), and severe cystic acne. In 2003 our group discovered that PSTPIP1 binds pyrin, the protein mutated in familial Mediterranean fever (FMF), and that disease-associated mutations in PSTPIP1 lead to more avid binding to pyrin, and increased IL-1 beta activation, relative to healthy controls. Progress During the Last Year Mechanism of inflammation in TRAPS: In May of this year we published a paper in PNAS that was the culmination of a several-year collaborative effort with Dr. Richard Siegel's group involving transfected cells, TRAPS knockin mice, and cells from TRAPS patients. Previous studies from our groups and others had shown that although there is the above-noted defect in ectodomain-cleavage of TRAPS-mutant TNF receptors, this is seen to a variable degree with different mutations and is cell-type dependent. Moreover, these mutant receptors bind TNF less well than wild-type receptors, and thus even if they do persist on the cell membrane after activation, it is not clear that retention of these receptor molecules would lead to repetitive stimulation, as we had originally hypothesized. Taken in the context of longer-term treatment failures with etanercept, the soluble p75:Fc fusion protein, in TRAPS, the pathogenesis of TRAPS has been an enigma. In the manuscript we published this year, we demonstrated that mutant TNFR1 accumulates intracellularly in peripheral blood mononuclear cells of TRAPS patients and in multiple cell types from two independent lines of knockin mice harboring TRAPS-associated TNFR1 mutations. Mutant TNFR1 did not function as a surface receptor for TNF but rather enhanced activation of MAPKs and secretion of proinflammatory cytokines upon stimulation with LPS. Enhanced inflammation depended on autocrine TNF secretion and WT TNFR1 in mouse and human myeloid cells but not in fibroblasts. Heterozygous TNFR1-mutant mice were hypersensitive to LPS-induced septic shock, whereas homozygous TNFR1-mutant mice resembled TNFR1-deficient mice and were resistant to septic shock. Thus WT and mutant TNFR1 act in concert from distinct cellular locations to potentiate inflammation in TRAPS. These findings establish a mechanism of pathogenesis in autosomal dominant diseases where full expression of the disease phenotype depends on functional cooperation between WT and mutant proteins and also may explain partial responses of TRAPS patients to TNF blockade. Gene expression profiling in CAPS: During the last year we completed a gene expression study in CAPS and have prepared a manuscript for submission. To understand the pathogenesis of CAPS we compared gene expression patterns in peripheral blool mononuclear cells (PBMCs) from 22 patients with active disease and 14 healthy children. We collected 16 paired samples from CAPS patients before and after anakinra (recombinant IL-1 receptor antagonist) treatment to identify transcripts responsive to IL-1 beta inhibition. We identified a gene expression signature that clearly distinguished CAPS patients from healthy controls. Many of the differentially expressed genes (DEGs) include transcripts related to regulation of innate and adaptive immune responses, oxidative stress, cell death, cell adhesion, and motility. Several DEGs, including transcripts related to regulation of reactive oxygen species (ROS), were validated by quantitative RT-PCR and by a functional study with primary cells from MWS patients and healthy controls. Using 17 CAPS samples and 66 non-CAPS samples, we created a set of gene expression-based models that differentiates CAPS patients from controls and from patients with other systemic inflammatory conditions. The CAPS-specific gene expression signature correctly classified all 17 samples from an independent dataset. This classifier also correctly identified 15 of 16 post-anakinra CAPS samples despite the fact that these CAPS patients were in clinical remission, thus validating the concept of gene expression profiling in the classification of inflammatory diseases and raising the possibility of IL-1 independent pathways in CAPS pathogenesis.
