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Familial Mediterranean Fever Mutations Lift the Obligatory Requirement for Microtubules in Pyrin Inflammasome Activation

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Familial Mediterranean Fever Mutations Lift the Obligatory Requirement for Microtubules in Pyrin Inflammasome Activation Familial Mediterranean fever mutations lift the obligatory requirement for microtubules in Pyrin inflammasome activation Hanne Van Gorpa,b,1, Pedro H. V. Saavedraa,b,1, Nathalia M. de Vasconcelosa,b, Nina Van Opdenboscha,b, Lieselotte Vande Wallea,b, Magdalena Matusiaka,b, Giusi Prencipec, Antonella Insalacoc, Filip Van Hauwermeirena,b, Dieter Demona,b, Delfien J. Bogaertd,e,f, Melissa Dullaersd,g, Elfride De Baereh, Tino Hochepieda,i, Joke Dehoornej, Karim Y. Vermaelenb,k, Filomeen Haerynckd,e,f, Fabrizio De Benedettic, and Mohamed Lamkanfia,b,2 aInflammation Research Center, VIB, Zwijnaarde, B-9052, Belgium; bDepartment of Internal Medicine, Ghent University, Ghent, B-9000, Belgium; cRheumatology Unit, Bambino Gesù Children’s Hospital, Rome, I-00146, Italy; dClinical Immunology Research Laboratory, Centre for Primary Immunodeficiency Ghent, Ghent University Hospital, Ghent, B-9000, Belgium; eDepartment of Pediatric Immunology and Pulmonology, Centre for Primary Immunodeficiency Ghent, Ghent University Hospital, Ghent, B-9000, Belgium; fJeffrey Modell Diagnosis and Research Centre, Ghent University Hospital, Ghent, B-9000, Belgium; gLaboratory of Immunoregulation, Inflammation Research Center, VIB, Zwijnaarde, B-9052, Belgium; hCenter for Medical Genetics Ghent, Ghent University, Ghent, B-9000, Belgium; iDepartment of Biomedical Molecular Biology, Ghent University, Ghent, B-9000, Belgium; jDepartment of Pediatric Rheumatology, Ghent University Hospital, Ghent, B-9000, Belgium; and kTumor Immunology Laboratory, Department of Pulmonary Medicine, Ghent University Hospital, Ghent, B-9000, Belgium Edited by Vishva M. Dixit, Genentech, San Francisco, CA, and approved October 28, 2016 (received for review August 8, 2016) Familial Mediterranean fever (FMF) is the most common monogenic (compound) heterozygous for mutations in MEFV, the gene that autoinflammatory disease worldwide. It is caused by mutations in the codes for the inflammasome adaptor Pyrin (4, 5). More than 310 inflammasome adaptor Pyrin, but how FMF mutations alter signaling disease-associated variants in MEFV have been reported to date in FMF patients is unknown. Herein, we establish Clostridium difficile in the InFevers registry (6), with most residing in the C-terminal and its enterotoxin A (TcdA) as Pyrin-activating agents and show that B30.2 (PRY/SPRY) domain of human Pyrin. Importantly, how- wild-type and FMF Pyrin are differentially controlled by microtubules. ever, how FMF mutations regulate Pyrin signaling has remained Diverse microtubule assembly inhibitors prevented Pyrin-mediated enigmatic, and mouse studies of FMF are complicated by the caspase-1 activation and secretion of IL-1β and IL-18 from mouse absence of the B30.2 domain in murine Pyrin. macrophages and human peripheral blood mononuclear cells FMF alleles occur in as many as one of every four individuals (PBMCs). Remarkably, Pyrin inflammasome activation persisted upon of non-Ashkenazi Jew, Arab, Armenian, and Turkish descent (7– microtubule disassembly in PBMCs of FMF patients but not in cells of 10). In addition, a subset of FMF patients is heterozygous for MEFV patients afflicted with other autoinflammatory diseases. We further disease-associated alleles, and the clinical/functional rel- MEFV demonstrate that microtubules control Pyrin activation downstream evance of some alleles is debated. Consequently, genetic of Pyrin dephosphorylation and that FMF mutations enable microtu- analysis of FMF is sometimes inconclusive, and FMF diagnosis bule-independent assembly of apoptosis-associated speck-like protein may be delayed for years (11). Although FMF is a systemic im- containing a caspase recruitment domain (ASC) micrometer-sized munological disease, immunological diagnosis of the disease is perinuclear structures (specks). The discovery that Pyrin mutations currently not available and is likely to require further insight into remove the obligatory requirement for microtubules in inflamma- how FMF mutations modulate Pyrin activation. The work presented some activation provides a conceptual framework for understanding FMF and enables immunological screening of FMF mutations. Significance FMF | Pyrin | inflammasome | colchicine | microtubules Familial Mediterranean fever (FMF) is an autoinflammatory dis- ease caused by more than 310 mutations in the gene MEFV,which nflammasomes are multiprotein complexes that culminate in encodes Pyrin. Pyrin recently was shown to trigger inflammasome processing of caspase-1, thereby promoting maturation of proIL- activation in response to Rho GTPase-modifying bacterial toxins. I Clostridium difficile 1β and proIL-18 into their active forms (1). Several inflammasome Here we report that infection and intoxication platforms have been described, and the concerted actions of with its enterotoxin TcdA engage the Pyrin inflammasome. More- inflammasomes frequently are of utmost importance for effective over, activation of the Pyrin inflammasome, but not other inflam- protection of the host against harmful environmental agents and masomes, was hampered by microtubule-depolymerizing drugs in infections (1). Conversely, mutations in genes coding for inflam- mouse and humans. Unexpectedly, we found that FMF mutations masome components and regulators cause debilitating systemic render Pyrin activation independent of microtubules. Thus, our autoinflammatory diseases, of which cryopyrin-associated periodic findings provide a conceptual framework for understanding Pyrin syndromes (CAPS; NLRP3 mutations), autoinflammation with signaling and enable functional diagnosis of FMF. infantile enterocolitis (AIFEC; NLRC4 mutations), hyperimmu- MVK Author contributions: H.V.G., P.H.V.S., and M.L. designed research; H.V.G., P.H.V.S., N.M.d.V., noglobulinemia syndrome (HIDS; mutations) and familial N.V.O., L.V.W., M.M., F.V.H., and D.D. performed research; G.P., A.I., D.J.B., M.D., E.D.B., T.H., Mediterranean fever (FMF; MEFV mutations) are notable ex- J.D., K.Y.V., F.H., and F.D.B. contributed new reagents/analytic tools; H.V.G., P.H.V.S., N.M.d.V., amples (2, 3). N.V.O., L.V.W., M.M., F.V.H., D.D., and M.L. analyzed data; H.V.G., P.H.V.S., and M.L. wrote FMF is the most common monogenic autoinflammatory dis- the paper; and M.L. coordinated the project. ease worldwide, affecting an estimated 150,000 patients (4). It Conflict of interest statement: H.V.G., P.H.V.S., and M.L. are listed as inventor on a patent typically has an autosomal recessive inheritance, and the clinical application on immunological FMF diagnosis. presentation is characterized by periodic fevers with childhood This article is a PNAS Direct Submission. onset, frequently accompanied by serositis and joint pain (3, 4). Freely available online through the PNAS open access option. The disease is highly prevalent in populations of the Eastern 1H.V.G. and P.H.V.S. contributed equally to this work. Mediterranean basin and the Middle East and has spread to the 2To whom correspondence should be addressed. Email: [email protected]. rest of the world with the extensive migrations of these pop- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ulations (4, 5). More than 80% of FMF patients are homo- or 1073/pnas.1613156113/-/DCSupplemental. 14384–14389 | PNAS | December 13, 2016 | vol. 113 | no. 50 www.pnas.org/cgi/doi/10.1073/pnas.1613156113 Downloaded by guest on October 6, 2021 here expands the set of Pyrin inflammasome-activating agents to bacteria and the expression of the bacterial toxins TcdA and TcdB live C. difficile infection and its enterotoxin TcdA. We further because inflammasome-dependent cytokine processing and secre- show that among the different known inflammasome sensors, tion were blunted when BMDMs were exposed to heat-killed wild-type Pyrin of both humans and mice relies selectively on C. difficile or were infected with the TcdA/B-deficient (VP11186) microtubules for inflammasome activation. Microtubules control C. difficile strain (Fig. S3 A and B). As with the purified toxins (Fig. Pyrin signaling downstream of Pyrin dephosphorylation. Sur- S1), C. difficile infection-induced caspase-1 activation required Pyrin prisingly, however, we found that FMF mutations lift the obligatory and the inflammasome adaptor ASC, whereas Nlrp3 and caspase- −/− −/− requirement for microtubules in activating the Pyrin inflamma- 11 were dispensable (Fig. S3 C and D). Likewise, Mefv and Asc some, providing a conceptual framework for understanding FMF BMDMs failed to secrete IL-1β in the culture supernatants, −/− and enabling immunological segregation of FMF from related whereas the supernatants of C. difficile-infected Nlrp3 and cas- −/− autoinflammatory disorders. pase-11 macrophages contained significant levels of IL-1β (Fig. S3 E and F). Notably, caspase-1 was responsible for the gross Results amount of IL-1β maturation and secretion, but proIL-1β maturation −/− and IL-1β secretion were not fully inhibited in caspase-1 caspase- TcdA Activates the Pyrin Inflammasome in Mouse Macrophages and −/− Human Monocytes. The Pyrin inflammasome responds to infection 11 macrophages (Fig. S3 C and E). These results suggest that with Burkholderia cenocepacia (12, 13) and the Rho GTPase- additional proteases may, to a limited extent, contribute to Pyrin- targeting toxins Clostridium botulinum toxin C3 and C. difficile and ASC-dependent IL-1β secretion in C. difficile-infected macro- cytotoxin B (TcdB) in mouse macrophages (13–15). Importantly, phages. Regardless, C. difficile-infected
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