During the current reporting period we have focused on 2 studies: 1) Regulation of the Pyrin Inflammasome Mutations in MEFV and MVK, encoding pyrin and mevalonate kinase (MVK), cause 2 distinct recessively inherited autoinflammatory diseases, familial Mediterranean fever (FMF) and hyperimmunoglobulinemia D syndrome (HIDS), respectively. IL-1beta plays an important role in the pathogenesis of both. Recently another group reported that pyrin forms an inflammasome, a multiprotein complex that mediates the maturation of IL-1beta by activating caspase-1, in response to bacterial modifications of RhoA. However, the molecular mechanism of pyrin inflammasome activation, as well as the molecular pathology of FMF and HIDS, remained unknown. In the current study, we found that the pyrin inflammasome is suppressed by pyrin phosphorylation through the RhoA signaling pathway, and that FMF and HIDS-associated mutations inhibit pyrin phosphorylation. Inactivation of RhoA triggers the pyrin inflammasome: The Clostridial TcdB and C3 toxins, which inactivate RhoA, activate IL-1beta maturation by a pathway that is Mefv-, Asc-, and Caspase-1-dependent, but Nlrp3-, Nlrc4-, and Aim2-independent. In contrast, IL-1beta secretion induced by the Clostridial toxins or from bone marrow-derived macrophages (BMDMs) of FMF knockin (KI) mice was substantially diminished when the BMDMs were treated with calpeptin, an indirect upstream RhoA activator or bacterial cytotoxic necrotizing factor (CNF) toxin, a direct RhoA activator. Another line of evidence supporting the inverse relationship between RhoA activation and pyrin inflammasome induction follows from our previous study of the inhibition of the NLRP3 inflammasome by intracellular cAMP, in which we observed that cAMP accentuates IL-1beta production by PBMCs from FMF patients. cAMP is known to suppress RhoA through phosphorylating RhoA. We observed a dose-dependent decrease of RhoA-GTP activity and an increase of IL-1beta secretion from the LPS-primed BMDMs of FMF-KI mice in response to adenylyl cyclase activation. These findings indicate that the pyrin inflammasome is activated by inactivation of RhoA. Since colchicine is a known activator for RhoA and an effective prophylaxis for FMF inflammatory attacks, the effect of colchicine on the pyrin inflammasome was also examined. Colchicine inhibited C3-toxin-induced IL-1beta secretion from LPS-primed BMDMs, as well as the constitutive IL-1beta secretion from BMDMs of FMF-KI mice and the PBMCs of FMF patients. The RhoA effector kinases, PKN1/2, suppress the pyrin inflammasome through pyrin phosphorylation and binding of 14-3-3 proteins: The inhibition of serine/threonine protein kinase C-related kinase (PKN/PRK), an effector kinase of RhoA, by inhibitors or gene knockdown induced pyrin-dependent IL-1beta secretion from LPS-primed BMDMs. Conversely, activation of PKNs with both bryostatin 1 and arachidonic acid substantially decreased C3-toxin induced IL-1beta secretion from LPS-primed BMDMs or the constitutive IL-1beta secretion from BMDMs of FMF-KI mice and from the PBMCs of FMF patients. In a coimmunoprecipitation assay, an interaction of pyrin with PKN1 was observed in lysates of LPS-primed BMDMs. The pyrin-PKN1 interaction was decreased by C3 toxin, but the decreased interaction was restored by cotreatment with arachidonic acid, indicating that activated PKN1 binds to pyrin and dissociates when PKN1 is inactivated. PKN1 interacts with pyrin through its C-terminal kinase domain and phosphorylates two serine residues (S208 and S242 of human pyrin). The phosphorylated pyrin binds 14-3-3 proteins, which block the pyrin inflammasome. The binding of 14-3-3 proteins as well as PKN1 to FMF-associated mutant pyrin is substantially less than binding to wild-type pyrin, and the constitutive IL-1beta secretion from FMF PBMCs or BMDMs of FMF-KI mice is attenuated by activating PKNs. HIDS is caused by activation of the pyrin inflammasome: The deficiency of MVK, a key enzyme in the mevalonate pathway, in HIDS or the pharmacological inhibition of HMG-CoA reductase results in the depletion of geranylgeranyl pyrophosphate, which leads to RhoA inactivation. Simvastatin, an inhibitor of HMG-CoA reductase, significantly decreased the 14-3-3 protein-pyrin interaction as well as the PKN1-pyrin interaction and induced pyrin inflammasome-dependent IL-1beta secretion from LPS-primed BMDMs. Simvastatin-induced IL-1beta secretion and the constitutive IL-1beta secretion from PBMCs of HIDS patients were blocked by activation of PKNs with arachidonic acid or bryostatin 1, which suppress the pyrin inflammasome. These findings indicate an essential role for the pyrin inflammasome in the pathogenesis of HIDS. We have submitted a manuscript describing these findings. 2) TRNT1-Associated Periodic Fever Syndrome Through whole-exome sequencing and candidate gene screening, we identified 5 children from 4 unrelated families with unexplained autoinflammatory disease and shared mutations in one common gene. All patients carried missense recessive mutations in TRNT1 (tRNA nucleotidyl transferase, CCA-adding, 1), encoded on chromosome 3. Two affected sisters from a Saudi Arabian consanguineous family were homozygous for a p.His215Arg missense mutation, while the other 3 children were compound heterozygous for a missense mutation, p.Ile223Thr or p.Arg99Trp, and one shared mutation, p.Asp163Val. p.His215Arg was not found in any public database nor in 1061 Arab control DNA samples. Among the 3 Caucasian mutations, p.Arg99Trp was novel whereas p.Ile223Thr and p.Asp163Val were found at a very low allele frequency (<0.001) in the NHLBI exome database. We subsequently identified 2 additional siblings who were compound heterozygous for p.Asp128Gly and p.Thr110Ile. p.Asp128Gly was found at a very low allele frequency (<0.001) in the ExAC database whereas p.Thr110Ile was novel. All mutations affect highly conserved amino acid residues and are predicted to be damaging to protein function. The TRNT1 protein adds CCA to the 3 prime end of all transfer RNAs both in the cytoplasm and mitochondria, which is critical for tRNA aminoacylation, protein synthesis, and tRNA degradation. Given the essential nature of this enzyme, disease-associated mutations are likely to be hypomorphs. All patients had recurrent episodes of fever and gastrointestinal symptoms with multisystem features that included developmental delay, nystagmus, hypotonia, optic nerve atrophy, sensorineural hearing loss, dysmorphic features, musculoskeletal symptoms, and B cell immunodeficiency. Multicolor flow cytometry of peripheral blood revealed an increased population of immature B cells, either naive B cells or transitional B cells. Flow cytometric studies suggested a B cell maturation defect in the bone marrow. Electron microscopy demonstrated degenerate mitochondria in various cell types, including cells of myeloid lineage and lymphocytes. Deep sequencing of tRNAs from patients fibroblasts showed a significant down-regulation of mature tRNAs when compared to healthy controls. RNA sequencing of patients whole blood revealed up-regulation of neutrophil-related genes. Consistent with these data, lesional biopsies from one patient's colon showed cryptitis with neutrophilic infiltration. Cytokine analyses in 2 patients have shown elevated levels of the proinflammatory cytokines interleukin 6 and IP-10, and to a lesser degree type 1 interferon. Unstimulated monocytes from 3 patients displayed an increased phosphorylation of STAT3 ex vivo. Knockdown of the zebrafish TRNT1 homolog caused hydrocephaly, defects in tail development, anemia, and a reduction in the number of hair cells present in the lateral line, which functions similarly to the human inner ear. A manuscript describing these findings is in preparation.

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Lee, Pui Y; Huang, Yuelong; Zhou, Qing et al. (2018) Disrupted N-linked glycosylation as a disease mechanism in deficiency of ADA2. J Allergy Clin Immunol 142:1363-1365.e8
Giannelou, Angeliki; Wang, Hongying; Zhou, Qing et al. (2018) Aberrant tRNA processing causes an autoinflammatory syndrome responsive to TNF inhibitors. Ann Rheum Dis 77:612-619
Meyts, Isabelle; Aksentijevich, Ivona (2018) Deficiency of Adenosine Deaminase 2 (DADA2): Updates on the Phenotype, Genetics, Pathogenesis, and Treatment. J Clin Immunol 38:569-578
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