Functional evaluation of the pathological significance of MEFV variants using induced pluripotent stem cell–derived macrophages
2019; Elsevier BV; Volume: 144; Issue: 5 Linguagem: Inglês
10.1016/j.jaci.2019.07.039
ISSN1097-6825
AutoresTakeshi Shiba, Takayuki Tanaka, Hiroaki Ida, Misa Watanabe, Haruna Nakaseko, Mitsujiro Osawa, Hirofumi Shibata, Kazushi Izawa, Takahiro Yasumi, Yuri Kawasaki, Megumu K. Saito, Junko Takita, Toshio Heike, Ryuta Nishikomori,
Tópico(s)IL-33, ST2, and ILC Pathways
ResumoFamilial Mediterranean fever (FMF) is the most common hereditary autoinflammatory disorder. It is characterized by recurrent episodes of fever, polyserositis, and abdominal pain. FMF is associated with mutations in the MEFV gene, which encodes the inflammasome adaptor pyrin. Pyrin is an inflammasome sensor that detects imbalances in Rho GTPase activity, which can be caused by bacterial effectors or bacterial toxins. Clostridial toxins are glycosylating enzymes that inactivate the RhoA GTPase, which results in activation of the pyrin inflammasome, activation of caspase-1, release of the proinflammatory cytokines IL-1β and IL-18, and an inflammatory cell death termed pyroptosis. More than 340 MEFV variants have been recorded in Infevers, an online database of autoinflammatory mutations. Among the MEFV variants, M694V and M694I in exon 10 were demonstrated in a systematic review to be related to a severe disease phenotype.1Gangemi S. Manti S. Procopio V. Casciaro M. Di Salvo E. Cutrupi M. et al.Lack of clear and univocal genotype-phenotype correlation in familial Mediterranean fever patients: a systematic review.Clin Genet. 2018; 94: 81-94Crossref PubMed Scopus (36) Google Scholar Other MEFV variants are associated with variable disease severity. The complexity of the clinical phenotype and its association with MEFV variants leads to difficulty in assessing the pathogenicity of variants identified in clinical settings. Successful use of IL-1β–blocking therapies in the treatment of FMF indicates that IL-1β plays a critical role in the pathogenesis of FMF.2De Benedetti F. Gattorno M. Anton J. Ben-Chetrit E. Frenkel J. Hoffman H.M. et al.Canakinumab for the treatment of autoinflammatory recurrent fever syndromes.N Engl J Med. 2018; 378: 1908-1919Crossref PubMed Scopus (238) Google Scholar Treatment with colchicine, a microtubule polymerization inhibitor, should be started as soon as a clinical diagnosis is made. However, in vitro pyrin inflammasome activation and its inhibition by colchicine in patients' hematopoietic cells remain controversial.3Chae J.J. Cho Y.H. Lee G.S. Cheng J. Liu P.P. Feigenbaum L. et al.Gain-of-function pyrin mutations induce NLRP3 protein-independent interleukin-1beta activation and severe autoinflammation in mice.Immunity. 2011; 34: 755-768Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar, 4Van Gorp H. Saavedra P.H. de Vasconcelos N.M. Van Opdenbosch N. Vande Walle L. Matusiak M. et al.Familial Mediterranean fever mutations lift the obligatory requirement for microtubules in Pyrin inflammasome activation.Proc Natl Acad Sci U S A. 2016; 113: 14384-14389Crossref PubMed Scopus (111) Google Scholar, 5Park Y.H. Wood G. Kastner D.L. Chae J.J. Pyrin inflammasome activation and RhoA signaling in the autoinflammatory diseases FMF and HIDS.Nat Immunol. 2016; 17: 914-921Crossref PubMed Scopus (325) Google Scholar, 6Jamilloux Y. Lefeuvre L. Magnotti F. Martin A. Benezech S. Allatif O. et al.Familial Mediterranean fever mutations are hypermorphic mutations that specifically decrease the activation threshold of the Pyrin inflammasome.Rheumatology (Oxford). 2018; 57: 100-111Crossref PubMed Scopus (42) Google Scholar In contrast to a previous report showing hypersecretion of IL-1β from blood cells derived from patients with FMF,3Chae J.J. Cho Y.H. Lee G.S. Cheng J. Liu P.P. Feigenbaum L. et al.Gain-of-function pyrin mutations induce NLRP3 protein-independent interleukin-1beta activation and severe autoinflammation in mice.Immunity. 2011; 34: 755-768Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar Van Gorp et al4Van Gorp H. Saavedra P.H. de Vasconcelos N.M. Van Opdenbosch N. Vande Walle L. Matusiak M. et al.Familial Mediterranean fever mutations lift the obligatory requirement for microtubules in Pyrin inflammasome activation.Proc Natl Acad Sci U S A. 2016; 113: 14384-14389Crossref PubMed Scopus (111) Google Scholar found that hypersecretion of IL-1β did not occur in monocytes from patients with FMF. To clarify this issue, we evaluated cytokine secretion from monocytes and monocyte-derived macrophages obtained from 5 patients with FMF carrying the M694I mutation (see Table E1 in this article's Online Repository at www.jacionline.org). The monocytes were stimulated with Clostridium difficile toxin A (TcdA) without priming with LPS because TcdA is sufficient to activate the pyrin inflammasome in monocytes4Van Gorp H. Saavedra P.H. de Vasconcelos N.M. Van Opdenbosch N. Vande Walle L. Matusiak M. et al.Familial Mediterranean fever mutations lift the obligatory requirement for microtubules in Pyrin inflammasome activation.Proc Natl Acad Sci U S A. 2016; 113: 14384-14389Crossref PubMed Scopus (111) Google Scholar and LPS induces IL-1β secretion through the NLRP3 inflammasome,7Gaidt M.M. Ebert T.S. Chauhan D. Schmidt T. Schmid-Burgk J.L. Rapino F. et al.Human monocytes engage an alternative inflammasome pathway.Immunity. 2016; 44: 833-846Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar complicating the evaluation of pyrin inflammasome activation. In response to TcdA stimulation, monocytes from patients with FMF secreted similar levels of IL-1β as monocytes from healthy donors (HDs) and asymptomatic carriers (ACs) expressing the E148Q variant (Fig 1, A). Colchicine inhibited IL-1β secretion from monocytes from HDs and ACs but not those from patients with FMF. We then examined inflammasome activation by evaluating apoptosis-associated speck-like protein containing a CARD (ASC) speck formation. Consistent with the IL-1β secretion, colchicine abolished TcdA-induced ASC speck assembly in monocytes from HDs and ACs but not those from patients with FMF (Fig 1, B, and see Fig E1 in this article's Online Repository at www.jacionline.org). Next, the IL-1β secretion from macrophages derived from monocytes in vitro (peripheral blood–derived macrophages [PB-MPs]) was evaluated. Because TcdA stimulation alone did not induce IL-1β secretion from PB-MPs (data not shown), PB-MPs were primed with LPS before TcdA stimulation. PB-MPs from patients with FMF secreted significantly greater levels of IL-1β than PB-MPs from HDs or ACs in response to stimulation with LPS and TcdA (Fig 1, C). The IL-1β secretion from PB-MPs from patients with FMF was inhibited by colchicine. These results suggest that although monocytes from patients with FMF are distinct in their unresponsiveness to colchicine inhibition, PB-MPs from patients with FMF show hyperactivation of the pyrin inflammasome, which is sensitive to colchicine inhibition. Thus, by examining both cell types from the same patients, we revealed the overall picture of the cytokine responses of monocytes and macrophages from patients with FMF. Induced pluripotent stem cell (iPSC) technology provides the opportunity to analyze the effect of genetic variants free from the influence of medication or differences in genetic background. Therefore we evaluated whether macrophages derived from patients' iPSCs (iPSC-derived macrophages [iPS-MPs]) recapitulated the phenotype of PB-MPs from patients with FMF. iPSC lines from 3 patients with FMF with the M694I and E148Q MEFV variants were established (patients 6-8; see Fig E2 and Table E1 in this article's Online Repository at www.jacionline.org) and differentiated into iPS-MPs (see Fig E3 in this article's Online Repository at www.jacionline.org). iPS-MPs with the M694I mutation recapitulated the enhanced pyrin inflammasome activation of PB-MPs, leading to increased IL-1β secretion (Fig 2, A), ASC speck formation (Fig 2, B), and cell death, which was dependent on MEFV expression (see Fig E4 in this article's Online Repository at www.jacionline.org). By contrast, the MEFV mutation did not affect NLRP3 inflammasome activation induced by LPS plus ATP stimulation. Although epidemiologic studies suggest that patients with FMF carrying MEFV exon 10 mutations, such as M694I, have a relatively early-onset severe clinical course,1Gangemi S. Manti S. Procopio V. Casciaro M. Di Salvo E. Cutrupi M. et al.Lack of clear and univocal genotype-phenotype correlation in familial Mediterranean fever patients: a systematic review.Clin Genet. 2018; 94: 81-94Crossref PubMed Scopus (36) Google Scholar the significance of other MEFV variants remains unknown. Therefore we applied our newly established method to 2 additional MEFV variants, T577N and N679H, which were identified in 2 families in which autoinflammatory disease with dominant inheritance was suspected. T577N was reported as a disease-causing variant in patients with attacks of fever and chest pain lasting longer than 1 week.8Stoffels M. Szperl A. Simon A. Netea M.G. Plantinga T.S. van Deuren M. et al.MEFV mutations affecting pyrin amino acid 577 cause autosomal dominant autoinflammatory disease.Ann Rheum Dis. 2014; 73: 455-461Crossref PubMed Scopus (78) Google Scholar By contrast, the patient carrying the N679H variant experienced typical FMF attacks.9Kishida D. Yazaki M. Nakamura A. Nomura F. Kondo T. Uehara T. et al.One novel and two uncommon MEFV mutations in Japanese patients with familial Mediterranean fever: a clinicogenetic study.Rheumatol Int. 2018; 38: 105-110Crossref PubMed Scopus (4) Google Scholar Consistent with the reported phenotype, no patient with the T577N variant in family 1 met the Tel-Hashomer criteria,E1Nakaseko H. Iwata N. Izawa K. Shibata H. Yasuoka R. Kohagura T. et al.Expanding clinical spectrum of autosomal dominant pyrin-associated autoinflammatory disorder caused by the heterozygous MEFV p.Thr577Asn variant.Rheumatology (Oxford). 2019; 58: 182-184PubMed Google Scholar whereas both patients with the N679H variant in family 2 met the criteria (see Fig E5 and Table E1 in this article's Online Repository at www.jacionline.org). Neither of the variants was registered in the Human Genetic Variation Database or the Exome Aggregation Consortium database, and both of the subsequent amino acid alterations were suggested to be disease causing in at least 1 of the 4 function-prediction programs used (see Table E2 in this article's Online Repository at www.jacionline.org). iPSC lines stably expressing wild-type (WT) MEFV or the E148Q, T577N, N679H, and M694I variants were generated (see Fig E5). iPS-MPs were derived from the genetically engineered iPSCs to evaluate pyrin inflammasome activation. Total and transgenic MEFV expression in iPS-MPs were comparable among the clones (see Fig E5). M694I iPS-MPs secreted significantly more IL-1β than E148Q or WT iPS-MPs (Fig 2, C). Although T577N iPS-MPs secreted comparable amounts of IL-1β to WT iPS-MPs, N679H iPS-MPs secreted significantly more IL-1β than WT iPS-MPs (Fig 2, C). ASC speck assembly was also enhanced in N679H and M694I iPS-MPs (Fig 2, D). These data indicate that, like the M694I variant, the N679H variant causes hyperactivation of the pyrin inflammasome. In summary, we characterized primary monocytes and macrophages from typical patients with FMF. The mechanism underlying the distinctive pyrin inflammasome activation in these cells remains to be elucidated. Gene expression differs considerably between monocytes and macrophages, including expression of the tubulin-related genes.E2Dong C. Zhao G. Zhong M. Yue Y. Wu L. Xiong S. RNA sequencing and transcriptomal analysis of human monocyte to macrophage differentiation.Gene. 2013; 519: 279-287Crossref PubMed Scopus (29) Google Scholar The greater expression of tubulin-related genes in macrophages might be related to the observation that pyrin inflammasome activation could be inhibited by the tubulin inhibitor colchicine in these cells. In addition, monocytes and macrophages are somewhat different in terms of inflammasome activation pathways. For example, although both cell types use the canonical NLRP3 inflammasome activation pathway, the alternative7Gaidt M.M. Ebert T.S. Chauhan D. Schmidt T. Schmid-Burgk J.L. Rapino F. et al.Human monocytes engage an alternative inflammasome pathway.Immunity. 2016; 44: 833-846Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar or noncanonicalE3Vigano E. Diamond C.E. Spreafico R. Balachander A. Sobota R.M. Mortellaro A. Human caspase-4 and caspase-5 regulate the one-step non-canonical inflammasome activation in monocytes.Nat Commun. 2015; 6: 8761Crossref PubMed Scopus (220) Google Scholar inflammasome activation pathway is functional only in monocytes. It is possible that an undiscovered pyrin inflammasome activation pathway is functioning in either monocytes or macrophages but not in both. After characterizing primary monocytes and macrophages from typical patients with FMF, we introduced a new method to functionally evaluate the pathological significance of MEFV variants by using iPS-MPs. Evaluation of diverse MEFV variants will be possible through this functional analysis and will facilitate our understanding of FMF pathogenesis. Macrophage dysfunction is observed in patients with other immune disorders. For example, macrophages are hyperactive in other autoinflammatory diseases, such as NLRC4-associated autoinflammatory diseases, whereas macrophage function is impaired in some immunodeficiencies, such as chronic granulomatous diseases. The current approach of iPSC genetic modification and efficient production of macrophages using iPSC-derived myeloid lineage cells (iPS-MLs) could be used to elucidate the pathogenesis of macrophage-related immune disorders other than FMF. We thank all participating patients, their families, and the referring physicians for their generous cooperation. Informed consent for genetic and functional analysis of blood samples was obtained from each patient or their guardians in accordance with the Declaration of Helsinki. The study was approved by the Ethics Committee of Kyoto University. Eight patients with FMF with the M694I mutation, 4 ACs with the E148Q variant, 9 HDs with no MEFV mutation, and 2 families with rare MEFV variants (T577N and N679H) were enrolled in this study (Table E1). All the participants were Japanese. All 8 patients with FMF with the M694I mutation fulfilled the Tel-Hashomer criteria.E4Livneh A. Langevitz P. Zemer D. Zaks N. Kees S. Lidar T. et al.Criteria for the diagnosis of familial Mediterranean fever.Arthritis Rheum. 1997; 40: 1879-1885Crossref PubMed Scopus (1252) Google Scholar PBMCs obtained from 5 patients with FMF (patients 1-5) were used for in vitro cytokine secretion assays, and PBMCs from the 3 other patients (patients 6-8) were used for establishing iPSCs. Two MEFV variants, T577N and N679H, were identified in families in which autoinflammatory disease with dominant inheritance was suspected through analysis of autoinflammatory disease–related genes (Table E3). The clinical course of family 1 is described elsewhere.E1Nakaseko H. Iwata N. Izawa K. Shibata H. Yasuoka R. Kohagura T. et al.Expanding clinical spectrum of autosomal dominant pyrin-associated autoinflammatory disorder caused by the heterozygous MEFV p.Thr577Asn variant.Rheumatology (Oxford). 2019; 58: 182-184PubMed Google Scholar Briefly, three patients experienced recurrent chest pain that lasted 3 to 6 months and was occasionally accompanied by a low-grade fever. Among the 3 symptomatic patients (I.1, II.1, and III.2), 1 (III.2) received colchicine treatment and had a favorable response. Two patients (II.2 and III.1) in family 2 experienced recurrent fever, unilateral pleuritis, and generalized peritonitis and responded well to colchicine treatment. Although no patient in family 1 met the Tel-Hashomer criteria, both patients in family 2 met the criteria (Table E1). PBMCs were isolated with Lymphoprep (Alere Technologies, Waltham, Mass). CD14+ monocytes were sorted magnetically from PBMCs by using the AutoMACS Pro Separator (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer's instructions. Monocytes were cultured in RPMI-1640 (Sigma-Aldrich, St Louis, Mo) supplemented with 10% FBS (Gibco, Carlsabad, Calif) and 50 ng/mL macrophage-colony stimulating factor (R&D Systems, Minneapolis, Minn) for 7 days to obtain monocyte-derived macrophages. Generation of iPSCs from patients with FMF with the M694I and E148Q variants (patients 6-8) was performed, as described previously.E5Nakagawa M. Taniguchi Y. Senda S. Takizawa N. Ichisaka T. Asano K. et al.A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells.Sci Rep. 2014; 4: 3594Crossref PubMed Scopus (394) Google Scholar Control iPSC lines were provided by the RIKEN BioResearch Center through the National Bio-Resource Project of the Ministry of Education, Culture, Sports, Science, and Technology, Japan. All iPSCs from patients with FMF carried the M694I and E148Q variants (Fig E2). After confirming the quality of the iPSCs, including marker gene expression, absence of residual vector expression, and pluripotency (Fig E2), iPS-ML lines were established by expressing MDM2, BMI1, and cMYC in monocytic lineage cells obtained from iPSCsE6Yanagimachi M.D. Niwa A. Tanaka T. Honda-Ozaki F. Nishimoto S. Murata Y. et al.Robust and highly-efficient differentiation of functional monocytic cells from human pluripotent stem cells under serum- and feeder cell-free conditions.PLoS One. 2013; 8: e59243Crossref PubMed Scopus (98) Google Scholar and then differentiated into macrophages (iPS-MPs). The mutant iPS-MLs and iPS-MPs showed comparable morphology and surface marker expression as control counterparts (Fig E3). piggyBac plasmids encoding WT or missense MEFV cDNA together with a blasticidin resistance gene (5 μg) and a plasmid encoding transposase (5 μg) were introduced into 1 × 106 control iPSCs with a NEPA21 electroporator (Nepa Gene, Ichikawa, Japan) to generate MEFV-reconstituted iPSCs. PiggyBac plasmids with missense MEFV variants were generated by using PCR-based mutagenesis with the primers listed in Table E4. Three days later, blasticidin (5 μg/mL; Wako Chemicals, Richmond, Va) was added to the medium for selection of MEFV-reconstituted cells. Genomic DNA and total RNA were extracted with the AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. Genomic DNA was diluted to 25 ng/mL in distilled water. cDNA was synthesized with PrimeScript RT Master Mix (Takara Bio, Shiga, Japan) from 500 ng of total RNA and diluted 1:10 in RNase-free water for analysis of OCT3/4 and NANOG mRNA expression. Expression of the pluripotent stem cell markers OCT3/4 and NANOG was confirmed by using TaqMan quantitative RT-PCR with the StepOnePlus Real-Time PCR System (Applied Biosystems/Thermo Fisher Scientific, Waltham, Mass). Primer and probe sequences are provided in Table E4. Expression of target genes was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in the same samples and expressed relative to expression in the 201B7 iPS cell line. Residual levels of the plasmids used for iPSC establishment were analyzed by using TaqMan quantitative PCR with the StepOnePlus Real-Time PCR System. Primer and probe sequences for CAG and EBNA1 were designed on the CAG promoter region and the EBNA1 cording region of all episomal vectors for iPSC generation, and they are listed in Table E4. Residual plasmid numbers were determined against a standard curve generated with known quantities of pCE-OCT3/4 episomal plasmid by using 50 ng of genomic DNA from iPSCs from patients with FMF at passage 3. Human pluripotent stem cells were differentiated into ectoderm, mesoderm, and endoderm lineages by using the STEMdiff Trilineage Differentiation Kit (STEMCELL Technologies, Vancouver, British Columbia, Canada). Human pluripotent stem cells reaching 70% to 80% confluency were harvested with TrypLE Select Enzyme (Thermo Fisher Scientific) and plated as a single-cell suspension in mTeSR1 medium (STEMCELL Technologies) containing 10 μmol/L Y-27632 (Wako Chemicals) on 6-well plates coated with Matrigel (BD Biosciences, San Jose, Calif). The cells were plated at 3.0 × 105 cells/well for differentiation into the ectoderm or mesoderm and 4.0 × 105 cells/well for differentiation into endoderm and harvested at day 7 (ectoderm) and day 5 (mesoderm and endoderm). Undifferentiated pluripotent stem cells and human pluripotent stem cell–derived ectoderm, mesoderm, and endoderm (1.0 × 106 cells each) were fixed with 4% paraformaldehyde in PBS (4% paraformaldehyde/PBS) for 20 minutes at 4°C and washed twice with staining medium that contained PBS with 2% FBS. Samples were permeabilized with BD Perm/Wash buffer (BD Biosciences) for 15 minutes at room temperature and stained for 1 hour with the fluorescently conjugated primary antibodies listed in Table E5. Samples were washed with BD Perm/Wash buffer twice and suspended in staining medium. Cells were acquired on an LSR flow cytometer (BD Biosciences). Data were analyzed and graphs were generated by using FlowJo software (FlowJo, Ashland, Ore). On day 3 of differentiation from iPS-MLs to iPS-MPs, 3.3 × 105 cells were transfected with 60 ng of Silencer Select predesigned small interfering RNA (siRNA; siRNA ID: s502555/s502557; Thermo Fisher Scientific) or Silencer Select Negative Control No. 1 siRNA using Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific), according to the manufacturers' instructions. Monocytes were seeded in flat-bottom 96-well plates (Falcon) at 5 × 104 cells/well in RPMI-1640 medium supplemented with 10% FBS. Colchicine (100 ng/mL; Sigma-Aldrich) was added, and cells were incubated for 30 minutes, followed by addition of 1 μg/mL TcdA (Enzo Life Sciences, Farmingdale, NY). After 4 hours of incubation, supernatants were collected. Macrophages were harvested with Accumax (Innovative Cell Technologies, San Diego, Calif) and seeded in 96-well plates. Colchicine was added, and cells were incubated for 30 minutes. After 2 hours of priming with 1 μg/mL LPS (InvivoGen, San Diego, Calif), 1 μg/mL TcdA was added, and supernatants were collected 4 hours later. For NLRP3 inflammasome activation, after priming iPS-MPs with LPS, nigericin (67 μmol/L; Sigma-Aldrich) or ATP (1 mmol/L; Sigma-Aldrich) was added, and the supernatants were collected 2 hours later. The IL-1β concentration was measured in duplicates by using the Bio-Plex Pro Human Cytokine Assay (Bio-Rad Laboratories, Hercules, Calif). Monocytes or macrophages were stimulated as in the cytokine secretion assay. After 2 hours of stimulation with TcdA, the cells were attached to slides with a Cytospin 4 Cytocentrifuge (Thermo Fisher Scientific), fixed in 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100 (Nacalai, Kyoto, Japan). Cells were incubated with an anti-ASC antibody (AL177; AdipoGen, San Diego, Calif) and then with an Alexa Fluor 594–labeled antibody to rabbit IgG (A11012; Invitrogen). Nuclei were stained with 4′,6-diamidino-2-phenylindole (Dojindo, Kumamoto, Japan). Cells were examined by using fluorescence microscopy under a BZ-X710 microscope (Keyence, Osaka, Japan), and BZ-X Analyzer software (Keyence) was used for quantitative analysis. Cells were seeded onto glass slides by using Platinum Pro (Matsunami, Bellingham, Wash) and stained with May-Grunwald and Giemsa staining solution (Merck Millipore, Burlington, Mass), according to the manufacturer's instructions. The slides were examined under a BZ-X710 microscope (Keyence), and the BZ-II Viewer software program (Keyence) was used for image acquisition. Expression of surface markers on hematopoietic cells was evaluated on a FACSVerse flow cytometer (BD Biosciences). Primary antibodies for analysis were as follows: CD14–allophycocyanin (Beckman Coulter, Fullerton, Calif) and phycoerythrin–Cy7-CD33 (BD Biosciences). Data analyses were performed with FlowJo software (TreeStar). Total RNA was extracted from iPS-MPs after stimulation with 1 μg/mL LPS for 4 hours by using the RNeasy Mini Kit (Qiagen). The RNA was subjected to DNase treatment and reverse transcribed into cDNA by using ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan). Real-time PCR was performed with TB Green Premix Ex TaqII (Tli RNaseH Plus; Takara Bio) and the StepOnePlus Real-Time PCR System, according to the manufacturers' instructions. The copy number of the target gene was determined against a standard curve of serial dilutions of the piggyBac vector. The primers used for detection of total or transgenic MEFV are listed in Table E2. Sytox Green permeability was used to quantify pyroptosis over time. iPS-MPs were seeded in duplicates at 1 × 104 cells per well in flat-bottom 96-well plates in the presence of 1.5 μmol/L Sytox Green (Invitrogen/Thermo Fisher Scientific). Cells were primed with LPS for 2 hours and then stimulated with 1 μg/mL TcdA. Data were acquired and analyzed with a fluorescent microscope (BZ-X710; Keyence) over a time span of 2.5 hours. Data are shown as mean ± SEMs, as indicated in figure legends. All statistical analyses were performed by using the Student t test or 1-way ANOVA with GraphPad Prism (version 8.00; GraphPad Software, La Jolla, Calif).Fig E2Characterization of iPSCs. A, Representative chromatogram of Sanger sequencing of MEFV. B, Quantitative RT-PCR validation of expression of the pluripotent stem cell–associated genes OCT3/4 and NANOG. Expression is shown relative to that of the 201B7 control clone. C, Quantification of the copy number of residual plasmids per cell. Copy numbers of the CAG promotor region and the EBNA1 sequence were determined. D, Flow cytometric evaluation of the in vitro differentiation capacity of control embryonic stem cells (H9 hESC), HD-derived iPSCs (Ct 1-3), and patient-derived lines (Pt 6-8).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Characterization of iPS-MLs and iPS-MPs. A, Representative May–Giemsa staining of iPS-MLs. Scale bars = 20 μm. B, Flow cytometric analysis of iPS-MLs. The white histogram represents the isotype control. C, Flow cytometric analysis of iPS-MPs. The white histogram represents the isotype control. D, Representative May–Giemsa staining of iPS-MPs. Scale bars = 20 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4Pyrin inflammasome activation in patient-derived iPS-MPs. A, Representative images of iPS-MPs. ASC, Red; nuclei, blue. Scale bars = 20 μm. B, iPS-MPs were stimulated with TcdA, and induction of pyroptosis was quantified over time by monitoring Sytox Green incorporation. *P < .05. C, Patient-derived iPS-MPs (n = 3) were transfected with MEFV siRNA (siRNA1 and siRNA2) or a scrambled control siRNA. Expression of MEFV was determined by using real-time PCR. Target gene levels were normalized to levels of ACTB, and results are expressed relative to levels in iPS-MPs transfected with scrambled siRNA. D, Patient-derived iPS-MPs (n = 3) transfected with scrambled control siRNA or MEFV siRNA were primed with LPS and then stimulated with TcdA (squares) or nigericin (triangles). IL-1β levels in the supernatant were assessed. ns, Nonsignificant.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E5Evaluation of rare MEFV variants using iPSC-MPs. A, Pedigrees of 2 families with rare MEFV variants: T577N (left) and N679H (right). B, Schematic diagrams of piggyBac vectors. C, Representative chromatogram of Sanger sequencing of MEFV with transgene-specific primers. D, A quantitative RT-PCR assay for expression of MEFV in genetically engineered iPSCs using a primer set that detects both the transgene and endogenous MEFV. E, Quantitative RT-PCR assay for expression of MEFV in genetically engineered iPSCs using a primer set that detects only expression of the MEFV transgene. Data are expressed as means ± SEMs of 4 independent experiments and were analyzed by using 1-way ANOVA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Genotypes and clinical characterization of patients with FMF and family control subjectsPatient IDMEFV genotypesInflammatory symptomsTel-Hashomer criteriaFMFPt 1Heterozygosity M694I and E148QYesYesPt 2Heterozygosity M694I and E148QYesYesPt 3Heterozygosity M694I and E148QYesYesPt 4Heterozygosity M694I and E148QYesYesPt 5Heterozygosity M694I, L110P, and E148QYesYesPt 6Heterozygosity M694I and E148QYesYesPt 7Heterozygosity M694I and E148QYesYesPt 8Heterozygosity M694I and E148QYesYesFamily 1I.1Heterozygosity T577N, homozygosity E148QYesNoII.1Heterozygosity T577N, homozygosity E148QYesNoII.2Heterozygosity E148QNoNoIII.2Heterozygosity T577N, homozygosity E148QYesNoFamily 2II.2Heterozygosity N679H, L110P, and E148QYesYesII.4Heterozygosity L110P, homozygosity E148QNoNoIII.1Heterozygosity N679H, L110P, and E148QYesYesIII.2Heterozygosity N679H and L110P, homozygosity E148QNoNoIII.3Heterozygosity E148QNoNo Open table in a new tab Table E2Functional predictions of the MEFV variants identified in families 1 and 2FamilyNucleotide changeAmino acid changeSIFTPolyPhen2Mutation TasterPROVEAN1c.1730C>Ap.Thr577AsnToleratedPossibly damagingPolymorphismNeutral2c.2035A>Cp.Asn679HisToleratedProbably damagingPolymorphismNeutral Open table in a new tab Table E3List of the autoinflammatory disease–related genes examinedPatient IDGenesHDs and ACsMEFV onlyFamily 1I.1, II.1, III.222 genes∗Twenty-two genes: CECR1, COPA, FAM105B, HMOX1, IL1RN, MEFV, MVK, NLRC4, NLRP12, NLRP3, NOD2, PLCG2, POMP, PSMA3, PSMB4, PSMB8, PSMB9, PSTPIP1, RBCK1, RNF31, TNFAIP3, and TNFRSF1A.II.2MEFV onlyFamily 2II.2, III.122 genes∗Twenty-two genes: CECR1, COPA, FAM105B, HMOX1, IL1RN, MEFV, MVK, NLRC4, NLRP12, NLRP3, NOD2, PLCG2, POMP, PSMA3, PSMB4, PSMB8, PSMB9, PSTPIP1, RBCK1, RNF31, TNFAIP3, and TNFRSF1A.II.4, III.2, III.3MEFV only∗ Twenty-two genes: CECR1, COPA, FAM105B, HMOX1, IL1RN, MEFV, MVK, NLRC4, NLRP12, NLRP3, NOD2, PLCG2, POMP, PSMA3, PSMB4, PSMB8, PSMB9, PSTPIP1, RBCK1, RNF31, TNFAIP3, and TNFRSF1A. Open table in a new tab Table E4List of primersForward primer sequenceReverse primer sequenceProbe sequenceProbe labelMutagenesis E148QCCTCCCGGCCTGGGGCTGGCTGCGCAGCCAGCCCCAGGCCGGGAGG T577NTCTGAACGCAGGTTTTCTGAGAAGTACTTTGTGCTCGAGCACAAAGTACTTCTCAGAAAACCTGCGTTCAGA N679HGGCGACAGAGTCATGTGCCCTTTCCTGCTTATGCATAAGCAGGAAAGGGCACATGACTCTGTCGCC M694IGGCTACTGGGTGGTGATAATGATAAAGGAAAATGAGTACCGGTACTCATTTTCCTTTATCATTATCACCACCCAGTAGCCVerification of transfected MEFV Template amplificationTGACCCTGCTTGCTCAACTCCTTCTCCCCTGTAGAAATGGTGCACAGGGCTAAGACAGTGACCCTGTTATCCCTAGCGGC Sequence reactionATTCCACACAAGAAAACGGCCTTCTCCCCTGTAGAAATGGTGCACAGGGCTAAGACAGTGGAAAGAGCAGCTGGCGAATGqPCR Total MEFVTGCACAGGGCTAAGACAGTGCATTTCTGAACGCAGGGTTT TransgeneTATCTGTCCAGTGGGTGGTCAGCACACCGGCCTTATTCCAA ACTBCACCATTGGCAATGAGCGGTTCAGGTCTTTGCGGATGTCCACGTiPSC characterization GAPDHTGCACCACCAACTGCTTAGCTCTTCTGGGTGGCAGTGATGACTCATGACCACAGTCCAVIC/MGB CAGGGCTCTGACTGACCGCGTTACAGAAAAGAAACAAGCCGTCATTTGTAATTAGCGCTTGGTTFAM/MGB EBNA1ATCAGGGCCAAGACATAGAGATGGCCAATGCAACTTGGACGTTTGTCCGGAGACCCCAFAM/MGB hOCT3/4Hs00999634_gHFAM/MGB hNANOGHs02387400_g1FAM/MGB Open table in a new tab Table E5List of antibodies for evaluation of iPSC differentiation into 3 germ layersAntibodiesCompanyCatalog no.DilutionApplicationOCT3/4–Alexa Fluor 647BD Biosciences, San Jose, Calif5603291:25Primary antibodyNANOG-FITCBD Biosciences5607911:25Primary antibodyPAX6-FITCBD Biosciences5616641:25Primary antibodySOX2-BV421BioLegend, San Diego, Calif6561141:50Primary antibodyBRACHYURY-PER&D Systems, Minneapolis, MinnIC2085P1:25Primary antibodyNCAM-BV421BioLegend3183281:25Primary antibodySOX17-Alexa647BD Biosciences5615891:25Primary antibodyFOXA2-PEBD Biosciences5625941:50Primary antibodyMouse IgG1,κ-APCBioLegend4001201:50Isotype controlMouse IgG1,κ–Alexa Fluor 488BioLegend4001291:25Isotype controlMouse IgG1,κ-PEBioLegend4001121:25Isotype controlMouse IgG1,κ-BV421BioLegend4001581:50Isotype controlAPC, Allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin. Open table in a new tab APC, Allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin.
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