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 disease 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 PBMCs of healthy individ- the B30.2 domain is absent in the murine Pyrin ortholog, which uals also secreted high levels of IL-1β and IL-18, and these prompted us to characterize regulation mechanisms of Pyrin acti- responses were inhibited by the cell-permeable caspase-1 inhibitor vation in peripheral blood mononuclear cells (PBMCs) of healthy Ac-YVAD-cmk (Fig. S3 G and H). As in murine macrophages, donors in parallel with studies in murine bone marrow-derived C. difficile-induced inflammasome activation in human PBMCs was macrophages (BMDMs). As reported (14, 15), TcdB activated the elicited by the bacterial toxins because IL-1β and IL-18 secretion Pyrin inflammasome in our studies (Fig. S1 A and B). C. difficile was blunted upon infection with the TcdA/TcdB-deficient produces a second enterotoxin, C. difficile toxin A (TcdA), which C. difficile mutant (Fig. S3 I and J). Together, these results dem- exerts markedly different functions in disease models (16). In ex- onstrate that TcdA and TcdB are fully responsible for C. difficile- amining the inflammasome response to TcdA, we found that induced inflammasome activation in rodents and humans alike. apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), but not caspase-11, was required for caspase-1 LPS Priming Is Dispensable but Enhances Pyrin Inflammasome Activation. maturation in LPS-primed BMDMs that had been incubated with Having established that the enterotoxins are required for inflam- TcdA (Fig. S1C). Also, proteolytic conversion of proIL-1β into the masome activation induced by C. difficile infection, we next set out mature cytokine required ASC and caspase-1 but not caspase-11 to investigate the mechanisms by which the enterotoxins engage the (Fig. S1 C and D). Consistent with TcdA activating a canonical Pyrin inflammasome. We first asked whether Pyrin activation re- inflammasome, extracellular release of IL-1β continued unabated quired priming, an important hallmark of the Nlrp3 inflammasome −/− in BMDMs lacking caspase-11 but was blunted in Asc macro- (23). Both TcdA and TcdB triggered caspase-1 maturation in −/− −/− phages, in caspase-1 caspase-11 BMDMs, and in macrophages unprimed macrophages, indicating that Toll-like receptor (TLR) of a newly generated caspase-1–deficient mouse strain (Fig. S1D priming is not a prerequisite for the Pyrin inflammasome (Fig. and Fig. S2). TcdA paralleled TcdB in engaging the Pyrin inflam- S4A). ProIL-1β is produced only in response to NF-κB cues such masome because TcdA-induced caspase-1 activation and subse- as TLR ligands, whereas unprimed BMDMs express a constitu- −/− quent cleavage of proIL-1β were abolished in Pyrin (Mefv ) tive pool of proIL-18 that is readily available for secretion upon −/− BMDMs but not in Nlrp3 macrophages (Fig. S1E). Consistent inflammasome activation (24). In agreement, culture medium of with these findings, TcdA-intoxicated macrophages required Pyrin, unprimed BMDMs secreted IL-18, but not IL-1β, when intoxicated but not Nlrp3, for secreting IL-1β (Fig. S1F). These results dem- with TcdA or TcdB (Fig. S4 B and C). As expected, LPS priming onstrate that TcdA selectively activates the Pyrin inflammasome in supported the secretion of mature IL-1β by the two toxins (Fig. murine BMDMs. To examine whether this selective activation also S4B) and further enhanced IL-18 secretion (Fig. S4C). Notably, INFLAMMATION

occurs in humans, PBMCs from three healthy donors were incu- LPS priming also markedly enhanced the upstream activation of IMMUNOLOGY AND bated with the cell-permeable caspase-1 inhibitor Ac-YVAD-cmk caspase-1 relative to levels seen in unprimed BMDMs (Fig. S4A). or the Nlrp3 inflammasome-selective inhibitor MCC950/CRID3 These results suggested that LPS may increase Pyrin expression in (17, 18) before intoxication with TcdA. Pharmacological inhibi- BMDMs. Indeed, both a meta-analysis of public (25–29) micro- tion of caspase-1 significantly reduced TcdA-induced cleavage of array data (Fig. S4D) and a longitudinal quantitative RT-PCR proIL-1β and the extracellular release of IL-1β and IL-18 from analysis of Pyrin expression levels showed that LPS increased Pyrin human PBMCs (Fig. S1 G–I). In contrast, TcdA-induced inflam- and Nlrp3 transcript levels by ∼10-fold in the first hours (Fig. S4 D masome activation was insensitive to MCC950/CRID3 at drug and E). At 6 h after LPS stimulation, Pyrin levels had increased concentrations that effectively blunted Nlrp3-relayed IL-1β and IL- further to ∼30-fold over baseline levels, whereas Nlrp3 remained 18 secretion from nigericin-treated PBMCs (Fig. S1 G–K). Thus, stable (Fig. S4E). Collectively, these findings indicate that, although our results confirm recent findings that purified TcdA selectively priming is not essential for Pyrin activation, it significantly enhances engages the Pyrin inflammasome in mouse BMDMs (19) and ex- Pyrin inflammasome activation by TcdA and TcdB. tend these results to human PBMCs. Microtubule-Depolymerizing Drugs Selectively Inhibit the Pyrin The Pyrin Inflammasome Is Engaged by C. difficile Infection. Both TcdA Inflammasome. Ectopically expressed Pyrin partially associates and TcdB contribute critically to C. difficile-induced pseudomem- with cytoskeletal structures (30), but how this localization relates branous colitis (16), although the pathogen also activates additional to its physiologic role in inflammasome activation is unclear. immune mechanisms independently of TcdA and TcdB (20). Be- We found that neither inhibition of actin polymerization with cause microbial pathogens may express several virulence factors cytochalasin D nor small-molecule targeting of downstream actin that engage multiple inflammasomes in parallel (21, 22), and be- effectors (c-Abl kinase with STI-571, myosin II with myostatin, cause the mechanisms of inflammasome activation induced by and Rock1/2 with Y-27632) interfered substantially with Pyrin- C. difficile infection are unknown, we next studied inflammasome induced IL-1β secretion from murine BMDMs and human responses in C. difficile-infected macrophages. Caspase-1 was acti- PBMCs (Fig. S5 A and B). Also the microtubule-stabilizing agent vated in wild-type BMDMs infected with C. difficile,andthisacti- paclitaxel (taxol) failed to modulate the secretion of IL-1β and vation resulted in substantial cleavage and extracellular release of IL-18 from TcdA-treated PBMCs (Fig. S5 C and D). In marked mature IL-1β (Fig. S3 A and B). These responses required live contrast, however, the microtubule polymerization inhibitor

Van Gorp et al. PNAS | December 13, 2016 | vol. 113 | no. 50 | 14385 Downloaded by guest on October 6, 2021 LPS+TcdA CYT997) also abolished the maturation of caspase-1, thereby

A BCD hampering the ensuing cleavage and secretion of IL-1β from TcdA-

treated BMDMs (Fig. 1 G and H). Paralleling these results, these tubulin polymerization inhibitors prevented Pyrin-dependent IL-1β

Untreated Mock Lumicolchicine Colchicine maturation in human PBMCs (Fig. 1I). Similarly, secretion of IL-1β Lumi-colchicine and IL-18 from TcdA-treated PBMCs was significantly reduced (Fig. 1 J and K), establishing that microtubules are essential for human and murine Pyrin activation. These findings led us to examine the role of microtubule polymerization in other inflam- Lumi-colchicine masomes. Anthrax lethal toxin engaged Nlrp1b-dependent auto- maturation of caspase-1 and cleavage of proIL-1β regardless of EF G H whether BMDMs had been pretreated with colchicine (Fig. 1L). Nlrc4-driven caspase-1 activation and intracellular IL-1β cleavage in Salmonella enterica serovar Typhimurium (S. Typhimurium)-infected macrophages also were normal in the presence of colchicine (Fig. 1M), as was activation of the AIM2 inflammasome by transfected dsDNA (Fig. 1N). Likewise, colchicine failed to modulate extracellular IL-1β release by each of these inflammasomes (Fig. S6

Lumi-colchicine Lumi-colchicine A–C). Nigericin-induced Nlrp3 inflammasome activation in murine BMDMs and human PBMCs was insensitive to colchicine in- I J K hibition (Fig. 1O and Fig. S6 D–F), and nigericin-induced caspase-1 activation along with downstream maturation and release of IL-1β continued unabated in the presence of the microtubule polymerization inhibitors nocodazole, ABT-751, CA4P, and CYT997 (Fig. S7). In conclusion, polymerized tubulin selectively and

LMNO AB C

Fig. 1. Microtubule depolymerizing drugs specifically inhibit the Pyrin in- DE flammasome. (A and B) Wild-type LPS-primed BMDMs were pretreated with lumicolchicine or colchicine before stimulation with TcdA. Samples were immunoblotted for caspase-1 and IL-1β (A), and supernatants were analyzed for IL-1β (B). (C) Unprimed BMDMs were pretreated with colchicine before infection with C. difficile followed by supernatant collection and analysis of IL-1β. (D–F) PBMCs from healthy donors (n = 3) were pretreated with lumicolchicine or colchicine before stimulation with TcdA. Samples were immunoblotted for IL-1β (D), and supernatants were analyzed for IL-1β (E)andIL-18(F). (G–K)LPS- primed BMDMs (G and H) and PBMCs from healthy donors (n = 3) (I–K) were pretreated with colchicine, nocodazole, ABT-751, CA4P, or CYT997 before F G stimulation with TcdA. Samples were immunoblotted for caspase-1 and IL-1β (G), and supernatants were analyzed for IL-1β (H). PBMC samples were immunoblotted for IL-1β (I), and supernatants were analyzed for IL-1β (J)andIL-18 (K). (L–O) LPS-primed BMDMs were pretreated with colchicine before being stimulated with activators of the Nlrp1b (anthrax lethal toxin; LeTx) (L), Nlrc4 (S. Typhimurium; STm) (M), AIM2 (dsDNA) (N), and Nlrp3 (nigericin; Nig) (O) inflammasomes followed by immunoblot to detect caspase-1 and IL-1β.Black arrowheads indicate procaspase-1 and proIL-1β, and white arrowheads indicate the p20 and p17 subunits. Luminex data are shown as mean ± SD,andalldata are representative of at least three independent experiments. Fig. 2. Differential microtubule regulation of Pyrin inflammasome activation identifies FMF patients. (A) PBMCs from healthy donors (n = 7) and FMF patients (n = 2) were infected with C. difficile, and the supernatant was analyzed for IL-1β. colchicine abolished caspase-1 maturation as well as downstream (B) PBMCs from healthy donors (n = 3) and FMF patients (n = 2) were pretreated cleavage and secretion of IL-1β from TcdA-treated BMDMs (Fig. 1 with Ac-YVAD-cmk before being stimulatedwithTcdA,andthesupernatantwas A and B). Lumicolchicine, a structurally related colchicine photo- analyzed for IL-1β.(C and D) PBMCs from healthy donors (n = 7) and FMF patients isomer that does not bind tubulin (31), did not affect Pyrin acti- (n = 2) were stimulated with LPS for 5 h (C) or were pretreated with colchicine A B before stimulation with TcdA (D), and the supernatant was analyzed for IL-1β.(E) vation (Fig. 1 and ), demonstrating the specificity of these = = results. Colchicine also abolished Pyrin-mediated IL-1β secretion PBMCs from healthy donors (n 3) and FMF patients (n 2) were pretreated C difficile C with colchicine, nocodazole, ABT-751, CA4P, or CYT997 before stimulation with from . -infected BMDMs (Fig. 1 ). Consistent with these TcdA, and the supernatant was analyzed for IL-1β.(F and G) PBMCs from healthy results, colchicine, but not lumicolchicine, inhibited Pyrin-induced = = = = β D donors (n 5) and from CAPS (n 4), JIA (n 7), and FMF (n 9) patients were proIL-1 maturation in human PBMCs (Fig. 1 ) and thus pre- pretreated with colchicine before stimulation with TcdA, and the supernatant vented the secretion of IL-1β and IL-18 from these cells (Fig. 1 E was analyzed for IL-1β (F)andIL-18(G). Luminex data are shown as mean ± SD, and F). Importantly, a set of structurally unrelated microtubule and all data are representative of at least three independent experiments. ns, polymerization inhibitors (nocodazole, ABT-751, CA4P, and non-significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

14386 | www.pnas.org/cgi/doi/10.1073/pnas.1613156113 Van Gorp et al. Downloaded by guest on October 6, 2021 ADB E

C F

Fig. 3. FMF-associated Pyrin binds tubulin but does not require microtubules for ASC speck assembly. (A) G I Schematic representation of different Flag-tagged Pyrin domain constructs. (B–D) Expression of different Flag-tagged Pyrin constructs in HEK293T cells followed by immunoprecipitation with Flag-beads and immunoblotting for tubulin and Flag. HEK293T cells were transfected with empty vector (EV), Flag- tagged wild-type (WT FL), or FMF (M694V) full- length human pyrin (FL) and its different domains, PYD, linker, PYD+linker and C-term, followed by immunoprecipitation with Flag-beads and immunoblotting for tubulin and Flag. (E and F) LPS-primed BMDMs (E) or HEK293T cells transfected with wild- type or FMF (M694V) full-length human pyrin (F) were pretreated with colchicine before stimulation with TcdA and then were immunoblotted to detect phosphorylated Pyrin (S241), IL-1β,andβ-actin (BMDMs), and phosphorylated Pyrin (S242), Flag, H J and tubulin (293T). PBMCs from healthy donors were pretreated with colchicine or CYT997 before stimulation with TcdA or nigericin (G and H). PBMCs from healthy donors and from CAPS, JIA, and FMF patients were pretreated with colchicine before stimulation with TcdA (I and J). ASC specks were analyzed by confocal microscopy as shown in mi- crographs (G and I) and automated quantification (H and J). Immunoblots and confocal images are rep- INFLAMMATION

resentative of at least three independent experiments. IMMUNOLOGY AND ns, non-significant; ***P < 0.001; ****P < 0.0001.

critically controls activation of the Pyrin inflammasome in humans blocked by the caspase-1 inhibitor Ac-YVAD-cmk (Fig. 2B). and rodents alike. Because murine Pyrin lacks the C-terminal Moreover, IL-1β levels secreted by FMF PBMCs in response to B30.2 domain of human Pyrin, these results also imply that this the NLRP3 inflammasome stimulus LPS (32, 33) were compara- region is dispensable for inflammasome activation and colchicine ble to those of healthy donors (Fig. 2C). These results indicate regulation. Nevertheless, the great majority of FMF mutations in that FMF mutations are not hypermorphic for inflammasome human Pyrin localize to the C-terminal B30.2 domain (4). activation relayed by either Pyrin or NLRP3. Moreover, they suggest that FMF mutations differ from CAPS-linked mutations in Differential Microtubule Regulation of Pyrin Inflammasome Activation NLRP3 that significantly enhance LPS- and cold-induced Nlrp3 Identifies FMF Patients. We therefore sought to determine how mi- inflammasome activation (33, 34). Remarkably, however, although crotubules relate to Pyrin inflammasome signaling in FMF PBMCs. colchicine pretreatment abolished TcdA-induced IL-1β secretion We reasoned that if microtubules relay an activating signal up- from PBMCs of healthy individuals (Fig. 2D), it augmented the stream of Pyrin, colchicine would halt TcdA-induced inflamma- TcdA-induced IL-1β secretion from FMF PBMCs (Fig. 2D). The some activation in PBMCs of FMF patients. To test this hypothesis, microtubule assembly inhibitors nocodazole, ABT-751, CA4P, and we collected PBMCs from seven healthy controls and two FMF CYT997 also had opposite influences on TcdA-induced IL-1β se- patients with confirmed MEFV mutations in the C-terminal B30.2 cretion from FMF PBMCs and from PBMCs of healthy donors domain [Table S1, patients FMF1 (M694V/R761H) and FMF2 (Fig. 2E). Furthermore, IL-18 release from TcdA-treated wild-type (M694I/M694I)]. We did not detect secreted IL-1β in culture PBMCs was blunted by colchicine and other microtubule poly- supernatants of untreated PBMCs of healthy donors or FMF merization inhibitors, whereas FMF PBMCs resisted suppression patients (Fig. 2A). C. difficile infection triggered a substantial but (Fig. S8). To validate these results further and to test whether comparable release of IL-1β in wild-type and FMF PBMCs (Fig. resistance to colchicine inhibition was a defining feature of FMF 2A). FMF PBMCs that had been intoxicated with TcdA also se- PBMCs, we repeated our studies with PBMCs from a larger group creted normal levels of IL-1β, a response that was efficiently of healthy donors (n = 5) and from an additional cohort of FMF

Van Gorp et al. PNAS | December 13, 2016 | vol. 113 | no. 50 | 14387 Downloaded by guest on October 6, 2021 patients with a variety of defined MEFV mutations (n = 9; Table polymerization inhibitor CYT997 (Fig. S10). Thus, FMF muta- S1). PBMCs from patients afflicted with CAPS disease resulting tions in Pyrin remove the critical dependency on microtubules for from heterozygous mutations in NLRP3 (n = 4; Table S1) and from ASC speck assembly and inflammasome activation downstream patients diagnosed with juvenile idiopathic arthritis (JIA, systemic of Pyrin dephosphorylation. and nonsystemic; n = 7) were tested also. As with PBMCs from healthy donors, colchicine blocked TcdA-induced IL-1β and IL-18 Discussion secretion from PBMCs of CAPS and JIA patients, indicating that The observation that Pyrin inflammasome activation by TcdA, their Pyrin inflammasome responses were regulated identically to TcdB, and live C. difficile infection required intact microtubules in those of healthy individuals (Fig. 2 F and G). In marked contrast, both human PBMCs and murine macrophages implies that the however, all FMF patients continued to secrete significant IL-1β C-terminal B30.2 domain, which harbors most FMF mutations in and IL-18 levels after colchicine pretreatment (Fig. 2 F and G). We humans but is absent in mouse Pyrin, is dispensable for inflamma- verified that colchicine efficiently disrupted assembled microtubules some activation. Although the B30.2 domain is dispensable for in the PBMCs of healthy individuals and FMF patients alike (Fig. Pyrin inflammasome activation, we established here that FMF S9), ruling out the remote possibility that microtubules of FMF mutations in this domain nonetheless remove the critical reliance on PBMCs resisted microtubule disassembly by colchicine. Together, intact microtubules for Pyrin-based nucleation of ASC specks and these results suggest that FMF mutations converge on lifting the crit- inflammasome signaling. Microtubules were recently proposed to ical requirement for microtubules in Pyrin inflammasome activation. control inflammasome activation apically of Pyrin dephosphorylation in response to bacterial RhoA inactivation (14). However, this FMF-Associated Pyrin Binds Tubulin but Does Not Require Microtubules suggestion is difficult to reconcile with the observation that TcdA- for ASC Speck Assembly. Dephosphorylation of Pyrin’s intermediate induced Pyrin dephosphorylation continued unhampered in linker domain was recently shown to be required for TcdA- and colchicine-pretreated macrophages and 293T cells (Fig. 3 E and TcdB-induced inflammasome activation (14, 15, 19), and intro- F and ref. 19). Moreover, we showed that FMF mutations render duction of FMF mutations in the B30.2 domain of ectopically Pyrin activation independent of microtubules. Thus, our results expressed Pyrin did not interfere with this process (15). In agreement provide a conceptual framework for understanding FMF based with these reports, we showed that endogenous Pyrin was not con- on a mechanistic model of Pyrin signaling in which microtubules stitutively active in FMF PBMCs but was engaged only after TcdA control inflammasome activation downstream of Pyrin dephos- stimulation or C. difficile infection (Fig. 2 and Fig. S8). To charac- phorylation (Fig. 4). In this model, microtubules relay an activating terize further how FMF mutations alter Pyrin-dependent inflam- signal to dephosphorylated wild-type Pyrin that shifts autorepressed masome activation, we examined the binding of ectopically expressed Pyrin into an open conformation. FMF mutations in the human Pyrin domains to endogenous tubulin (Fig. 3A). Flag-fused con- B30.2 domain may force dephosphorylated Pyrin in an open constructs corresponding respectively to the N-terminal PYD domain, formation that readily binds the inflammasome adaptor ASC, ef- the intermediate linker sequence, the PYD+linker combination, or fectively replacing microtubule-relayed signals (Fig. 4). the C-terminal tripartite motif (TRIM) and B30.2 domains were Paradoxically, although we show here that FMF mutations render expressed in 293T cells and were immunoprecipitated with Flag- Pyrin inflammasome activation insensitive to colchicine, this drug is beads. Constructs containing either the PYD or the C-terminal re- an effective treatment that suppresses periodic inflammatory attacks gions of wild-type Pyrin coprecipitated endogenous tubulin, but the in the majority of FMF patients and prevents amyloidosis, a major intermediate linker sequence did not (Fig. 3B). This result suggests long-term complication of the disease that may result in renal failure that Pyrin interacts with tubulin through both its N and C termini. and death (5, 36). However, the clinical efficacy of colchicine treat- However, introduction of the FMF-associated M694V mutation did ment is likely associated with its ability to decrease leukocyte motility C not prevent tubulin binding in either full-length Pyrin (Fig. 3 )orin and phagocytosis during inflammation (37, 38). Recent studies sug- D the isolated carboxyl-terminal region (Fig. 3 ). As reported (19), gested that defects in the mevalonate pathway seen in the hereditary TcdA also triggered Pyrin dephosphorylation in wild-type BMDMs autoinflammatory disease mevalonate kinase deficiency (MKD), also E that had been pretreated with colchicine (Fig. 3 ). Furthermore, named “hyperimmunoglobulinemia D syndrome” (HIDS), may also colchicine failed to prevent TcdA-induced dephosphorylation trigger unwarranted activation of the Pyrin inflammasome (14, 39). of ectopically expressed wild-type Pyrin and the FMF-associated M694V Pyrin mutant in 293T cells (Fig. 3F). In agreement with our other studies showing that microtubule polymerization inhibitors prevented inflammasome activation selectively in wild- C. difficile TcdA type, but not in FMF, PBMCs, this finding positions microtubules in the pathway downstream of Pyrin dephosphorylation. RhoA glucosylation PYD-based inflammasome sensors such as Pyrin nucleate ASC filaments through a biphasic mechanism in which their PYD domain first nucleates ASC PYD to oligomerize into prion-like fi- Pyrin Ser208/Ser242 dephosphorylation bers that then condense into ASC specks through ASC–caspase recruitment domain (CARD) interactions that also enable the Wildtype FMF recruitment of caspase-1 zymogens (35). Interference with these Pyrin Pyrin first and second stages of ASC speck assembly therefore results in either the complete absence or the formation of atypical fila- microtubules microtubules mentous fibers, respectively (35). We exposed PBMCs of healthy donors to TcdA or nigericin to trigger ASC speck assembly through the Pyrin and NLRP3 inflammasomes, respectively (Fig. 3 G and Inflammasome assembly H). Colchicine and the unrelated microtubule polymerization inhibitor CYT997 selectively prevented ASC speck formation in response to TcdA but not in nigericin-treated PBMCs (Fig. 3 G and pro-IL-1β IL-1β H), demonstrating that microtubules were selectively required for pro-IL-18 IL-18 nucleation of ASC specks by Pyrin but not by NLRP3. As in healthy Fig. 4. Schematic model of Pyrin inflammasome activation by RhoA-modifying donors, colchicine abolished TcdA-induced ASC speck assembly toxins. C. difficile TcdA and TcdB inactivate RhoA, thereby triggering de- in PBMCs of CAPS and JIA patients (Fig. 3 I and J). In marked phosphorylation of Pyrin and its release from inhibitory 14-3-3 proteins. Mi- contrast, however, the assembly of ASC specks by FMF PBMCs crotubules are critical for activation of wild-type Pyrin in human PBMCs and was unabated in the presence of colchicine (Fig. 3 I and J). murine macrophages. In contrast, FMF-associated mutations in Pyrin render ASC Similar results were obtained with the unrelated microtubule speck assembly and inflammasome activation independent of microtubules.

14388 | www.pnas.org/cgi/doi/10.1073/pnas.1613156113 Van Gorp et al. Downloaded by guest on October 6, 2021 In contrast to FMF patients, however, MKD patients generally do Materials and Methods not benefit from colchicine treatment, whereas blockade of IL-1 has All reported patients and healthy controls provided written informed consent shown promising results (40). Given our observations that FMF for participation in the study, in accordance with International Conference mutations render Pyrin activation resistant to colchicine blockade, it on Harmonization of Technical Requirements for Registration of Pharmaceu- would be interesting to investigate the role of microtubules in MKD- ticals for Human Use/Good Clinical Practice (ICH/GCP) guidelines. The research associated inflammasome activation. protocol was approved by the ethics committee of Ghent University Hospital Akin to colchicine, certain pathogens express toxins that manip- under number 2012_593 and the protocols of Bambino Gesù Children’s Hospital. ulate microtubule dynamics, as exemplified by the CDT toxin of All mice were kept in specific pathogen-free conditions within the animal hypervirulent C. difficile strains (41). It therefore is tempting to speculate facilities of Ghent University. All animal experiments were approved by the that the high frequency of heterozygous MEFV mutations in endemic FMF regions (42) might be related to their rendering Pyrin acti- ethics committee on laboratory animal welfare of Ghent University. vation insensitive to microtubule manipulations by such pathogens. Detailed methods used in all experiments throughout this work are de- Given the key role of inflammasomes in antimicrobial host defense scribed in SI Materials and Methods. (43), the ability to engage the Pyrin inflammasome in the presence of microtubule dynamics blockade is likely to have offered het- ACKNOWLEDGMENTS. We thank the patients and their families who provided specimens for this study; Vishva Dixit and Nobuhiko Kayagaki (Genentech) for erozygous individuals a selective advantage in clearing such in- generously supplying mutant mice; and Amelie Fossoul (VIB-University of Ghent) fections. Finally, the insight that inflammasome activation by FMF and the VIB Bio Imaging Core for technical support. F.V.H., L.V.W., and N.V.O. Pyrin resists colchicine blockade enables functional/immunological are postdoctoral fellows with the Fund for Scientific Research-Flanders. This work screening of the disease among clinically overlapping autoin- was supported by Ghent University Concerted Research Actions Grant BOF14/ flammatory patients and thus may contribute to timely diagnosis GOA/013, European Research Council Grant 281600, and a Baillet Latour Medical and commencement of therapy in the future. Research Grant (to M.L.).

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