During the current reporting period we have made progress in four thematic areas: (1) the cell biology of TRAPS;(2) long-term follow-up of a cohort of TRAPS patients treated with the TNF-inhibitor etanercept;(3) regulation of the NLRP3 inflammasome;and (4) discovery of a new dominantly inherited disorder of inflammation. Cell Biology of TRAPS Because reactive oxygen species (ROS) have been shown to play an important role in normal inflammatory responses and other autoinflammatory diseases, we hypothesized that ROS might also play a role in the abnormal signaling and cytokine production in cells harboring TNFR1 mutations. In this collaborative study with Richard Siegels group we found that ROS generated by mitochondrial respiration are in fact important for normal lipopolysaccharide (LPS)-driven production of several proinflammatory cytokines and for the enhanced responsiveness to LPS seen in patients with TRAPS. We found elevated baseline ROS in both mouse embryonic fibroblasts and human immune cells harboring TRAPS-associated TNFR1 mutations. A variety of antioxidants dampened LPS-induced MAP kinase phosphorylation and inflammatory cytokine production. However, gp91phox and p22phox reduced nicotinamide adenine dinucleotide phosphate (NADPH) subunits were dispensable for inflammatory cytokine production, indicating that NADPH oxidases are not the source of proinflammatory ROS. TNFR1 mutant cells exhibited altered mitochondrial function with enhanced oxidative capacity and mitochondrial ROS generation, and pharmacologic blockade of mitochondrial ROS efficiently reduced cytokine production after LPS stimulation in cells from TRAPS patients and healthy controls. These findings suggest that mitochondrial ROS may be a novel therapeutic target for TRAPS and other inflammatory diseases. This work was published earlier this year in the Journal of Experimental Medicine. Long-Term Follow-up of a Cohort of TRAPS Patients Treated with Etanercept When we first described TRAPS in 1999, we found impaired activation-induced shedding of TNF receptors from patients leukocytes. Receptor-shedding is thought to have a negative homeostatic effect, because it reduces the density of membrane TNF receptors available for immune cell activation, and because shed receptors bind TNF in solution and thereby inhibit its binding to membrane receptors. We reasoned that treatment with the recombinant TNFR1:Fc fusion protein etanercept might restore homeostasis. Although our initial experience with etanercept was gratifying, we subsequently observed partial responses in some patients, and the emerging body of cell biologic data suggests that blocking TNF would be unlikely to block the inflammatory manifestations of TRAPS completely. The present study provides a long-term analysis of 15 patients with TRAPS enrolled in a prospective, open-label dose-escalation study and followed for 10 years. In the initial phase of the study, the patients were observed for three months at baseline, three months on 50 mg etanercept subcutaneously per week (or 0.8 mg/kg sq weekly for children), three months on 75 mg etanercept sq weekly (or 1.2 mg/kg sq weekly for children), and for three months in a washout phase. Patients recorded attacks, symptom severity, and use of ancillary medications in a daily diary. Blood samples were collected during each period and measured for acute phase reactants. Seven to nine years after conclusion of the initial study, patients completed a follow-up servey 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, pateints 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. These data are consistent with the hypothesis that TNF is only one part of the pathophysiology of TRAPS. A manuscript summarizing these results is in press in Arthritis and Rheumatism. Regulation of the NLRP3 Inflammasome Mutations in the gene encoding NLRP3 cause a spectrum of autoinflammatory disease known as the cryopyrin-associated periodic syndromes (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. ATP, a well-known activator for the NLRP3 inflammasome, triggers potassium efflux through the P2X7 receptor (P2X7R). Although it has been suggested that intracellular potassium efflux is critical, the precise molecular mechanism of NLRP3 inflammasome activation, as well as CAPS-associated mutant NLRP3 activation, remains to be elucidated. In this project we showed that the calcium sensing receptor (CaSR) is essential for NLRP3 inflammasome activation, mediated by increased intracellular calcium and decreased cAMP. Without ATP, calcium or CaSR agonists activated the NLRP3 inflammasome, whereas knockdown of the CaSR reduced inflammasome activation. Subsequently, we identified phospholipase C (PLC), which produces inositol triphosphate and results in the release of calcium from endoplasmic reticulum (ER) storage, to be a downstream mediator of the CaSRs activation of the NLRP3 inflammasome. The increased cytoplasmic calcium promoted the assembly of inflammasome components. We also found that cyclic AMP (cAMP) binds to NLRP3 to suppress inflammasome activation. The binding affinity of cAMP with CAPS-associated mutant NLRP3 is substantially lower than the wild-type NLRP3, and the uncontrolled mature IL-1 production from CAPS patients peripheral blood mononuclear cells (PBMCs) was attenuated by increasing cAMP. Taken together, these data suggest that cAMP is a direct regulator of the NLRP3 inflammasome and an essential component in the molecular pathogenesis of CAPS. A manuscript describing these findings is currently under review. Discovery of 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 family. 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. A manuscript describing this work is currently under review.

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