The current reporting period marks the culmination of five major studies in this thematic area. Each resulted in a paper that was published or accepted for publication over the last year. Long-Term Follow-up of a Cohort of TRAPS Patients Treated with Etanercept This study provides a long-term analysis of 15 patients with TRAPS enrolled in a prospective, open-label dose-escalation study of the TNFR1:Fc fusion protein etanercept and followed for 10 years. In the initial phase of the study, the patients were observed for 3 months at baseline, 3 months on 50 mg etanercept subcutaneously per week (or 0.8 mg/kg sq weekly for children), 3 months on 75 mg etanercept sq weekly (or 1.2 mg/kg sq weekly for children), and for 3 months in a washout phase. Patients recorded attacks, symptom severity, and use of ancillary medications in a daily diary. Acute phase reactants were measured during each period. Seven to nine years after conclusion of the initial study, patients completed a follow-up survey and were evaluated to determine the long-term outcome of etanercept treatment. Etanercept significantly attenuated the total symptom score and reduced the frequency of symptoms in the initial phase. Etanercept also reduced acute phase reactants, particularly during asymptomatic periods. During the 10-year follow-up period, patients remained on etanercept for a median of 3.3 years, with a number of patients switching to anti-IL-1beta therapy or remaining off biologic agents, citing injection site reactions and lack of efficacy most frequently for discontinuation. However, patients remaining on etanercept had reduced symptoms at follow-up. The data are consistent with the hypothesis that TNF is only one part of the pathophysiology of TRAPS. Gene Expression Profiling in CAPS Patients Treated with Anakinra The goal of this study was to define a gene expression profile that distinguishes CAPS patients from controls, and to assess the effectiveness of anakinra in normalizing this profile. We collected peripheral blood mononuclear cells (PBMCs) from 22 CAPS patients with active disease and 14 healthy children. Transcripts that passed stringent filtering criteria (false discovery rate of less than 1%) were considered differentially expressed genes (DEGs). A set of DEGs was validated by quantitative RT-PCR and functional studies with primary cells from CAPS patients and healthy controls. We used 17 CAPS and 66 non-CAPS patient samples to create a set of gene expression models that differentiates CAPS patients from controls and from patients with other autoinflammatory conditions. Many of the DEGs are related to the regulation of innate and adaptive immune responses, oxidative stress, cell death, cell adhesion, and motility, and were in common with other systemic inflammatory diseases such as systemic-onset juvenile idiopathic arthritis. A set of gene expression-based models comprising 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 patients despite the fact that these CAPS patients were in clinical remission, suggesting incomplete suppression of inflammation at these doses of anakinra. Regulation of the NLRP3 Inflammasome Mutations in the NLRP3 cause CAPS. The NLRP3 inflammasome is one of several cytoplasmic multiprotein complexes that mediate the maturation of the proinflammatory cytokine interleukin-1beta by activating caspase-1. The mechanism of NLRP3 inflammasome activation, as well as the mechanism by which CAPS-associated mutations activate NLRP3, remain to be elucidated. In this project we showed that the calcium sensing receptor (CaSR) activates the NLRP3 inflammasome, mediated by increased intracellular calcium and decreased cyclic AMP (cAMP). Calcium or other CaSR agonists activate the NLRP3 inflammasome in the absence of exogenous ATP, whereas knockdown of CaSR reduces inflammasome activation in response to known NLRP3 activators. The CaSR activates the NLRP3 inflammasome through phospholipase C (PLC), which catalyzes inositol triphosphate production and thereby induces release of calcium from endoplasmic reticulum stores. The increased cytoplasmic calcium promotes the assembly of inflammasome components, and intracellular calcium is required for spontaneous inflammasome activity in cells from CAPS patients. CaSR stimulation also results in reduced intracellular cAMP, which independently activates the NLRP3 inflammasome. cAMP binds to NLRP3 directly to inhibit inflammasome assembly, and downregulation of cAMP relieves this inhibition. The binding affinity of cAMP for CAPS-associated mutant NLRP3 is substantially lower than for wild-type NLRP3, and the uncontrolled mature IL-1beta production from CAPS patients PBMCs is attenuated by increasing cAMP. Taken together, these findings suggest that calcium and cAMP are key molecular regulators of the NLRP3 inflammasome that have critical roles in the molecular pathogenesis of CAPS. Discovery of PLAID, a New Dominantly Inherited Disorder of Inflammation In collaboration with Dr. Josh Milner, we identified 3 families with a dominantly inherited complex of cold urticaria, antibody deficiency, and susceptibility to infection and autoimmunity. Cold-induced urticaria occurred in all affected individuals. Other variable manifestations included atopy, granulomatous rash, autoimmune thyroiditis, antinuclear antibodies, sinopulmonary infections, and common variable immune deficiency. Serum IgM and IgA, and circulating NK and class-switched memory B cells, were reduced. B cell receptor-editing, NK cell degranulation, and ligand-induced calcium flux in both cell types were impaired, while enhanced cellular function in mutant PLCG2-expressing cells was observed at subphysiologic temperatures. Linkage analysis demonstrated a 7 Mb candidate interval on chromosome 16q (LOD = 4.2) in one family, overlapping by 3.5 Mb a disease-associated haplotype in a smaller family. This interval includes PLCG2, encoding a signaling molecule expressed in B, NK, and mast cells. cDNA sequencing revealed heterozygous transcripts lacking exon 19 in 2 families, and exons 20-22 in the third. Genomic sequencing identified 3 distinct in-frame deletions that co-segregated with disease. These deletions, located within an autoinhibitory domain, produce protein products with constitutive phospholipase activity. We have proposed the acronym PLAID (phospholipase C gamma2-associated antibody deficiency and immune dysregulation) to denote this condition. Discovery of APLAID, an Autoinflammatory Disease Caused by a Hypermorphic Missense Mutation in PLCG2 Whole-exome sequencing was performed in a family affected by dominantly inherited inflammatory disease characterized by recurrent blistering skin lesions, bronchiolitis, arthralgia, ocular inflammation, enterocolitis, absence of autoantibodies, and mild immunodeficiency. Exome data from 3 samples, including the affected father and daughter and unaffected mother, were filtered for the exclusion of reported variants, along with benign variants, as determined by PolyPhen-2. A total of 8 transcripts were identified as possible candidate genes. We confirmed a variant, p.S707Y, within PLCG2 as the only de novo variant that was present in 2 affected family members and not present in 4 unaffected members. The substitution is located in the same autoinhibitory domain harboring deletions in PLAID. Overexpression of S707Y protein and ex vivo experiments using affected individuals leukocytes showed clearly enhanced PLCG2 enzyme activity and evidence of increased intracellular signaling even at physiologic temperatures. We have proposed the acronym APLAID (autoinflammatory PLAID) to denote this clinically and immunologically distinct disease.
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