During the current reporting period we focused on 3 studies: 1) Regulation of the Pyrin Inflammasome Many of the salient findings of this project were summarized in last years report. Briefly, we confirmed the observation that bacterial toxins that inactivate RhoA activate the pyrin inflammasome. We went on to demonstrate that RhoA effector kinases PKN1/2 phosphorylate human pyrin at residues 208 and 242, leading to the binding of 14-3-3 proteins to pyrin and inhibition of pyrin inflammasome assembly. Inhibition of RhoA prevents the phosphorylation of pyrin and releases pyrin-bound 14-3-3, allowing the assembly of the pyrin inflammasome. During the previous reporting period we also found that colchicine, an established treatment for familial Mediterranean fever (FMF), activates RhoA and thus blocks the pyrin inflammasome, and that inhibition of mevalonate kinase, as is seen in the hyperimmunoglobulinemia D with periodic fever syndrome, inactivates RhoA by blocking its localization to the inner cell membrane and thereby leads to pyrin inflammasome activation. We also demonstrated that FMF-associated mutations in pyrin are associated with impaired binding of PKN and 14-3-3 proteins to pyrin, thus accounting for the lower threshold for pyrin inflammasome activation in FMF. During the current reporting period we published 2 papers in Nature Immunology describing these findings. During the current reporting period we also collaborated with Dr. James Bliska at Stony Brook University to test the hypothesis that toxins produced by Yersinia species might impinge on the pyrin inflammasome activation pathway. Pathogenic Yersina, including Y. pestis, the agent of plague in humans, and Y. pseudotuberculosis, the related enteric pathogen, deliver virulence effectors into host cells via a prototypical type III secretion system to promote pathogenesis. These effectors, termed Yersinia outer proteins (Yops), modulate multiple host signaling responses. Studies in Y. pestis and Y. pseudotuberculosis have shown that YopM suppresses infection-induced inflammasome activation; however, the underlying molecular mechanism was largely unknown. In this series of experiments we showed that YopM specifically restricts the pyrin inflammasome, which is triggered by the RhoA-inactivating enzymatic activities of YopE and YopT, in Y. pseudotuberculosis-infected macrophages. The attenuation of a yopM mutant is fully reversed in pyrin knockout mice, demonstrating that YopM inhibits pyrin to promote virulence. Mechanistically, YopM recruits and activates PKN1/2 to negatively regulate pyrin by phosphorylation. These results show how a virulence factor can hijact host kinases to inhibit effector-triggered pyrin inflammasome activation. During the current reporting period we published a paper in Cell Host and Microbe describing these findings. 2) Discovery of Otulipenia, a New Autoinflammatory Disease We recently discovered a new autoinflammatory disease caused by loss-of-function mutations in FAM105B, encoding the linear deubiquitinase OTULIN. We identified 1 frameshift and 2 missense mutations in 1 Pakistani and 2 Turkish families with 4 affected patients. Patients presented with neonatal-onset fever, neutrophilic dermatitis/panniculitis, and failure to thrive, but without obvious primary immunodeficiency. HEK293 cells transfected with mutated FAM105B had decreased enzyme activity relative to cells transfected with WT FAM105B, and showed a substantial defect in the linear deubiquitination of target molecules. Stimulated patients fibroblasts and peripheral blood mononuclear cells showed evidence for increased signaling in the canonical NF-kappaB pathway and accumulated linear ubiquitin aggregates. Levels of proinflammatory cytokines were significantly increased in the stimulated primary cells and serum samples. This discovery adds to the emerging spectrum of human diseases caused by defects in the ubiquitin pathway and suggests a role for targeted cytokine therapies. During the current reporting period a manuscript describing these findings was published in the PNAS. 3) Examination of the Effectiveness of Tumor Necrosis Factor (TNF) Inhibitors in the Deficiency of Adenosine Deaminase 2 (DADA2) In the FY2014 reporting period we published a paper in the New England Journal of Medicine describing DADA2, a recessively inherited disorder characterized by recurrent fevers, livedo racemosa, early-onset lacunar strokes, and, in some patients, polyarteritis nodoasa (PAN). It is caused by mutations in CECR1, encoding ADA2. In a retrospective analysis of 15 patients referred to our clinic in whom we made the diagnosis of DADA2 and who had been treated with a wide spectrum of potent immunosuppressives (but not TNF inhibitors), patients had an average of 1 stroke every 21 months. Based on our previous demonstration of perivascular TNF in DADA2 biopsy specimens and the preliminary favorable response of Israeli DADA2 patients with PAN to TNF inhibitors, we compared the frequency of stroke in 15 NIH DADA2 patients before and after initiation of TNF inhibitors (etanercept, infliximab, adalimumab). Before anti-TNF initiation, these 15 patients had 55 strokes over 1173 patient months (model based recurrence rate 0.043) compared to 0 strokes over 373 patient months after initiation of anti-TNF (recurrence rate 0; P = 1.68 x 10e-8). A matched follow up time analysis using 373 patient months both prospectively and retrospectively estimates the probability of stroke before initiation of anti-TNF as opposed to afterward to be 1 (Blyth-Still-Casella exact confidence interval = 0.7873, 1; P = 5.1 x 10e-5). TNF inhibition was also associated with significant improvement in laboratory parameters. We also examined the frequency of hemorrhagic strokes in DADA2 patients prior to their referral to the NIH, stratified by whether they had received aspirin and/or anticoagulants. The proportions of patients with hemorrhagic strokes on no antiplatelet or anticoagulant treatment, aspirin alone, or aspirin plus other anticoagulants and/or antiplatelet agents, were 0/9, 0/6, and 4/7, respectively (P = 0.007, Fischers exact test). Our data strongly support the use of TNF inhibitors with a history of stroke, but argue against the use of aspirin plus other anticoagulants in these patients. We are currently preparing a manuscript describing these findings.

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Human Genome Research
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Oda, Hirotsugu; Kastner, Daniel L (2017) Genomics, Biology, and Human Illness: Advances in the Monogenic Autoinflammatory Diseases. Rheum Dis Clin North Am 43:327-345
Demirkaya, Erkan; Zhou, Qing; Smith, Carolyne K et al. (2017) Brief Report: Deficiency of Complement 1r Subcomponent in Early-Onset Systemic Lupus Erythematosus: The Role of Disease-Modifying Alleles in a Monogenic Disease. Arthritis Rheumatol 69:1832-1839
Netea, Mihai G; Balkwill, Frances; Chonchol, Michel et al. (2017) A guiding map for inflammation. Nat Immunol 18:826-831
Manthiram, Kalpana; Zhou, Qing; Aksentijevich, Ivona et al. (2017) The monogenic autoinflammatory diseases define new pathways in human innate immunity and inflammation. Nat Immunol 18:832-842
Stoffels, Monique; Kastner, Daniel L (2016) Old Dogs, New Tricks: Monogenic Autoinflammatory Disease Unleashed. Annu Rev Genomics Hum Genet 17:245-72
Park, Yong Hwan; Wood, Geryl; Kastner, Daniel L et al. (2016) Pyrin inflammasome activation and RhoA signaling in the autoinflammatory diseases FMF and HIDS. Nat Immunol 17:914-21
Ozen, Seza; Demirkaya, Erkan; Erer, Burak et al. (2016) EULAR recommendations for the management of familial Mediterranean fever. Ann Rheum Dis 75:644-51
Chung, Lawton K; Park, Yong Hwan; Zheng, Yueting et al. (2016) The Yersinia Virulence Factor YopM Hijacks Host Kinases to Inhibit Type III Effector-Triggered Activation of the Pyrin Inflammasome. Cell Host Microbe 20:296-306
Akula, Murali K; Shi, Man; Jiang, Zhaozhao et al. (2016) Control of the innate immune response by the mevalonate pathway. Nat Immunol 17:922-9
Brehm, Anja; Liu, Yin; Sheikh, Afzal et al. (2015) Additive loss-of-function proteasome subunit mutations in CANDLE/PRAAS patients promote type I IFN production. J Clin Invest 125:4196-211

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