Gain-of-function CEBPE mutation causes non-canonical autoinflammatory inflammasomopathy

Gain-of-function CEBPE mutation causes noncanonical autoinflammatory inflammasomopathy Helka G€ o€ os, MSc, a * Christopher L. Fogarty, PhD, b,c,d,e * Biswajyoti Sahu, PhD, f * Vincent Plagnol, PhD, g Kristiina Rajam€ aki, PhD, h Katariina Nurmi, PhD, h Xiaonan Liu, MSc, a Elisabet Einarsdottir, PhD, i,j,k Annukka Jouppila, MSc, l Tom Pettersson, MD, PhD, h,m Helena Vihinen, DSc, n Kaarel Krjutskov, PhD, j,l,o P€ aivi Saavalainen, PhD, p,q Asko J€ arvinen, MD, PhD, r Mari Muurinen, MD, i,k Dario Greco, PhD, a,s Giovanni Scala, PhD, a,s James Curtis, PhD, t Dan Nordstr€ om, MD, PhD, h,u Robert Flaumenhaft, MD, PhD, v Outi Vaarala, MD, PhD, w,x Panu E. Kovanen, MD, PhD, y Salla Keskitalo, PhD, a Annamari Ranki, MD, PhD, z Juha Kere, MD, PhD, i,j,k,aa Markku Lehto, PhD, b,c,d Luigi D. Notarangelo, MD, bb Sergey Nejentsev, MD, PhD, t Kari K. Eklund, MD, PhD, h,u,cc àMarkku Varjosalo, PhD, a à Jussi Taipale, PhD, f,dd,ee àand Mikko R. J. Sepp€ anen, MD, PhD r,ff àHelsinki and Tampere, Finland; London and Cambridge, United Kingdom; Stockholm, M€ olndal, and Solna, Sweden; Tartu, Estonia; Boston, Mass; and Bethesda, Md GRAPHICAL ABSTRACT Background: CCAAT enhancer–binding protein epsilon (C/EBPε) is a transcription factor involved in late myeloid lineage differentiation and cellular function. The only previously known disorder linked to C/EBPεis autosomal recessive neutrophil-specific granule deficiency leading to severely impaired neutrophil function and early mortality. Objective: The aim of this study was to molecularly characterize the effects of C/EBPεtranscription factor From a the Institute of Biotechnology, HiLIFE, d the Diabetes & Obesity Research Program, Research Program’s Unit, e the Institute of Clinical Medicine, f the Research Programs Unit, Genome-Scale Biology, Biomedicum Helsinki, h Clinicum, Faculty of Medicine, k the Research Programs Unit, Molecular Neurology, n the Electron Microscopy Unit, Institute of Biotechnology, p the Research Programs Unit, Immunobiology, and q the Department of Medical and Clinical Genetics, University of Helsinki; b the Folkh€ alsan Research Center, Helsinki; c Abdominal Center Nephrology, r the Adult Immunodeficiency Unit, Infectious Diseases, Inflammation Center, u the Department of Rheumatology, Inflammation Center, w the Pediatric Research Center, z the Department of Dermatology, Allergology and Venereal Diseases, Inflammation Center, and ff the Rare Diseases Center and Pediatric Research Center, Children’s Hospital, University of Helsinki and Helsinki University Hospital; g University College London Genetics Institute, University College London; i Folkh€ alsan Institute of Genetics, Helsinki; j the Department of Biosciences and Nutrition, Karolinska Institutet, Stockholm; l Helsinki University Hospital Research Institute; m the Department of Internal Medicine and Rehabilitation, Helsinki University Hospital, Helsinki; o the Competence Centre on Health Technologies, Tartu; s the Faculty of Medicine and Life Sciences & Institute of Biosciences and Medical Technology, University of Tampere; t the Department of Medicine, University of Cambridge; v Beth Israel Deaconess Medical Center, Department of Medicine, Harvard Medical School, Boston; x Respiratory, Inflammation and Autoimmunity, Innovative Medicine, AstraZeneca, M€ olndal; y the Department of Pathology, University of Helsinki, and HUSLAB, Helsinki University Hospital; aa the School of Basic and Medical Biosciences, King’s College London, Guy’s Hospital, London; bb the Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda; cc Orton Orthopaedic Hospital and Research Institute, Invalid Foundation, Helsinki; dd the Division of Functional Genomics and Systems Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Solna; and ee the Department of Biochemistry, Cambridge University. *These authors contributed equally to this work. àThese authors contributed equally to this work. 1364 Arg219His mutation identified in a Finnish family with previously genetically uncharacterized autoinflammatory and immunodeficiency syndrome. Methods: Genetic analysis, proteomics, genome-wide transcriptional profiling by means of RNA-sequencing, chromatin immunoprecipitation (ChIP) sequencing, and assessment of the inflammasome function of primary macrophages were performed. Results: Studies revealed a novel mechanism of genome-wide gain-of-function that dysregulated transcription of 464 genes. Mechanisms involved dysregulated noncanonical inflammasome activation caused by decreased association with transcriptional repressors, leading to increased chromatin occupancy and considerable changes in transcriptional activity, including increased expression of NLR family, pyrin domain-containing 3 protein (NLRP3) and constitutively expressed caspase-5 in macrophages. Conclusion: We describe a novel autoinflammatory disease with defective neutrophil function caused by a homozygous Arg219His mutation in the transcription factor C/EBPε. Mutated C/EBPεacts as a regulator of both the inflammasome and interferome, and the Arg219His mutation causes the first human monogenic neomorphic and noncanonical inflammasomopathy/immunodeficiency. The mechanism, including widely dysregulated transcription, is likely not unique for C/EBPε. Similar multiomics approaches should also be used in studying other transcription factor–associated diseases. (J Allergy Clin Immunol 2019;144:1364-76.) Key words: Immunologic deficiency syndromes, autoinflammatory diseases, hereditary, chemotaxis, interferons, inflammasomes, NLR family, pyrin domain-containing 3 protein, gain-of-function mutation, neomorphic mutation Primary immunodeficiencies (PIDs) are caused by inherent defects in the immune system and offer a unique glimpse into regulation of the human immune system. Findings in patients with PIDs can help in development of treatments for immune dysregulation, which is known to lead to various common diseases, including atherosclerosis, asthma, and diabetes. 1 Of the more than 350 currently known PIDs, approximately 20 are caused by germline autosomal dominant gain-of-function (GOF) mutations. 2-4 In general, GOF mutations can be caused by hyperactivating or neomorphic mechanisms. To date, all known GOF PIDs have been hypermorphic (ie, resulting in increased protein activity). 4 No neomorphic mutations resulting in completely novel molecular functions have been described in patients with PIDs. Many autoinflammatory diseases (AIDs), such as cryopyrinopathies, are driven by activation of the canonical NLR family, pyrin domain-containing 3 protein (NLRP3) inflammasome through production of the highly proinflammatory IL-1band IL-18. The noncanonical caspase-4/5 (murine ortholog, caspase-11) inflammasome is a recently discovered inflammasome activated by intracellular bacterial LPS and involved in first-line defense against gram-negative bacteria. Similar to canonical activation, noncanonical inflammasome activation results in increased IL-1band IL-18 production. 5 In mice induction of caspase-11 expression by type I interferon signaling is required for activation of the noncanonical inflammasome. 6-9 In human subjects local activation of the noncanonical inflammasome has been shown to contribute to the pathogenesis of age-related macular degeneration. 10 However, the potential role of the noncanonical inflammasome in systemic human diseases remains to be explored. The CCAAT enhancer–binding protein epsilon (C/EBPε), encoded by CEBPE, is a transcription factor expressed in myeloid and lymphoid lineage cells and is known to be involved in cellular differentiation and function of late myeloid lineages. 11 The only Abbreviations used AID: Autoinflammatory disease CAIN: C/EBPε-associated autoinflammation and immune impairment of neutrophils C/EBP: CCAAT enhancer–binding protein ChIP: Chromatin immunoprecipitation ChIP-seq: ChIP, Chromatin immunoprecipitation sequencing FC: Fold change FDR: False discovery rate GOF: Gain of function JAK: Janus kinase LOF: Loss of function NF-kB: Nuclear factor kB NLRP3: NLR family, pyrin domain-containing 3 protein PID: Primary immunodeficiency PPI: Protein-protein interactions RNA-seq: RNA sequencing SGD: Neutrophil-specific granule deficiency SMRC2: SWI/SNF complex subunit SMARCC2 STAT: Signal transducer and activator of transcription WT: Wild-type This study was supported by the Orion Research Foundation (to H.G.); the Paulo Foundation ja Maire Lisko Foundation (to K.R.); Helsinki University Hospital Research funds (to A.R. and M.R.J.S.), an Academy of Finland Post-doctoral Fellowship (274555, to B.S.); the P€ aivikki and Sakari Sohlberg Foundation and the Yrj€ o Jahnsson Foundation (to K.N.); the Division of Intramural Research, National Institute of Health, National Institutes of Health, Bethesda, Maryland (to L.D.N.); the Folkh€ alsan Research Foundation and the Novo Nordisk (to M.L.); the Academy of Finland (288475 and 294173, to M.V.); the Sigrid Jus elius Foundation (to M.V.); the Finnish Foundation for Pediatric Research (to M.R.J.S.); a University of Helsinki Three-year Research Grant, Biocentrum Helsinki, Biocentrum Finland, and HiLIFE (to M.V.); the Instrumentarium Research Foundation (to M.V. and K.K.E.); and Finska L€ akares€ allskapet (to K.K.E. and D.N.). Disclosure of potential conflict of interest: The authors declare that they have no relevant conflicts of interest. Received for publication November 2, 2018; revised May 6, 2019; accepted for publication June 4, 2019. Available online June 13, 2019. Corresponding author: Mikko R. J. Sepp€ anen, MD, PhD, Rare Diseases Center and Pediatric Research Center, Children’s Hospital, University of Helsinki and Helsinki University Hospital, PO Box 281, FI-00029 HUS Helsinki, Finland. E-mail: mikko. [email protected]. The CrossMark symbol notifies online readers when updates have been made to the article such as errata or minor corrections 0091-6749 Ó2019 The Authors. Published by Elsevier Inc. on behalf of the American Academy of Allergy, Asthma & Immunology. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). https://doi.org/10.1016/j.jaci.2019.06.003 J ALLERGY CLIN IMMUNOL VOLUME 144, NUMBER 5 G € O € OS ET AL 1365 previously known CEBPE-associated disorder, autosomal recessive neutrophil-specific granule deficiency (SGD), is caused by frameshift mutations resulting in abrogation of at least 2 of the 4 known C/EBPεisoforms. This leads to a complete loss of specific neutrophilic granules and consequently to severely impaired neutrophil function with pronounced susceptibility to bacterial infections and early mortality. 12,13 A milder autosomal dominant CEBPE (Val218Ala) variant leads to total SGD with recurrent deep-seated abscesses. 14 Early studies in Cebpe knockout mice showed Cebpe to be integral for maintenance of constitutive levels of several cytokines, including IFN-g. 15 We report an autosomal recessive GOF PID, C/EBPε-associated autoinflammation and immune impairment of neutrophils (CAIN), in a family with a genetically uncharacterized autoinflammatory syndrome. 16-18 According to our results, C/EBPεacts as a regulator of both the inflammasome and interferome. Homozygous missense mutations in CEBPE (14:23586886 C->T;p.Arg219His) resulted in markedly decreased C/EBPεassociation with transcriptional repressors and increased occupancy on chromatin, leading to dysregulated C/EBPε-mediated transcription of interleukin and interferon response genes in neutrophils. Patients’ macrophages displayed aberrant caspase-5–mediated activation of the noncanonical inflammasome pathway. Patients with CAIN display a loss-of-function (LOF) mechanism in protein-protein interactions (PPIs) and a novel GOF mechanism in DNA binding and transcriptional regulation. Furthermore, CAIN is the first PID and systemic AID involving a noncanonical inflammasome. METHODS Written informed consent was obtained from study participants, and the study was approved by the institutional ethical review board (138/13/03/00/ 2013). DNA from 3 affected family members (Fig 1,A) were analyzed by using whole-exome sequencing, and Sanger sequencing was further used to confirm the mutation status of the other family members. Before whole-exome sequencing was available, patients were tested against LBR,MVK, and NLRP3 mutations; no rare cosegregated variants in known AID genes were noted. Further details of the genetic analysis are available in the Methods section in this article’s Online Repository at www.jacionline.org. Functional studies of CEBPE mutation Flp-In T-REx 293 cell lines stably expressing mutant or wild-type (WT) C/EBPεwere generated and used to investigate PPIs by using proximitydependent biotin identification coupled to mass spectrometry, as described by Liu et al 19 and in the Methods section in this article’s Online Repository. Functional studies of patients’ primary cells Granulocytes were isolated from 2 homozygous patients, 2 heterozygous carriers, and 3 sexand age-matched control subjects, and chromatin immunoprecipitation sequencing (ChIP-seq) was used to assess C/EBPε binding to chromatin in freshly isolated and LPS-stimulated cells. RNA sequencing (RNA-seq) was used for genome-wide transcriptional profiling of freshly isolated untreated granulocytes from patients and control subjects. Given the clinical phenotype of increased bacterial infections, we also used 59-end RNA-seq to identify genes differentially expressed after 3 different stimulations: (1) DNA extracted from Pseudomonas aeruginosa, (2) LPS extracted from P aeruginosa, or (3) LPS extracted from Escherichia coli. Moreover, granulocytes were stimulated with IFN-a2 and IFN-g, followed by transcriptional profile analysis using 59-end RNA-seq. Nanostring 20 analysis was used for direct digital detection of the mRNA levels of selected genes from PBMCs. Caspase-5 levels were analyzed by using quantitative PCR and Western blotting to access inflammasome activation and inflammasome-mediated cytokine secretion more deeply. Caspase-1 activity was measured by using flow cytometry from blood immune cells, and IL-1b/IL-18 secretion was measured by using ELISA from cultured macrophages. We also assessed granule exocytosis, granulocyte responsiveness, and neutrophilic and monocytic nuclear factor kB (NF-kB) phosphorylation using flow cytometry. Subcellular morphology of granulocytes and granule abundance of platelets were assessed by using transmission electron microscopy, as well as Wright staining of granulocytes. Further details of the methods are available in the Method section in this article’s Online Repository. RESULTS Case reports In the 1970s, affected members of the index family were thought to have atypical Pelger-Hu€ et anomaly because they presented with neutrophil hyposegmentation, aberrant neutrophil responsiveness, and impaired chemotaxis. 16-18 Patients experienced recurrent attacks of abdominal pain, aseptic fever, and systemic inflammation lasting 4 to 5 days. These were accompanied by an acute-phase response and occasionally by nailbed, tongue, submandibular and gluteal abscesses; intra-abdominal granulomas; pyoderma gangrenosum; and buccal ulcerations. Furthermore, they experienced frequent episodes of purulent paronychia complicated by lymphangitis, superficial skin, and mucosal and purulent upper respiratory tract infections. Their autoinflammatory symptoms manifested more clearly during puberty and subsided after menopause (Table I). All affected members further had mild bleeding diathesis with frequent nosebleeds and a tendency toward hematomas after needle sticks and procedures. Extended case reports are available in this article’s Online Repository at www.jacionline.org. Genetic analysis found a novel homozygous CEBPE mutation To identify the causative mutation, we analyzed DNA from 3 surviving members (II.2, II.7, and II.13) of the index family by performing whole-exome sequencing. In exome data we identified between 20,638 and 21,313 single nucleotide variants and small insertions/deletions in each patient. A total of 149 variants were very rare (ie, those not seen in the 6500 National Heart Lung, and Blood Institute Exomes 1000 Genomes database [April 2012 data release] and 2500 exomes analyzed internally by using the same bioinformatics pipeline). Of these, 22 variants were shared between all 3 patients. Only one of these variants was homozygous in all 3 patients (ENSG00000092067:ENST00000206513: exon2:c.G656A;p.Arg219His) in the CEPBE gene. The novel homozygous CEBPE 14:23586886 C>T mutation cosegregated perfectly with the disease phenotype (Fig 1,A, and see Fig E1 in this article’s Online Repository at www.jacionline.org). 16-18 Arg219His was predicted to be detrimental and not listed in major public or in-house databases. It resided in the highly conserved basic zipper region’s carboxyl terminal DNA-binding domain shared by all 4 C/EBPεisoforms (Fig 1,Band C). 11,21,22 Identified homozygous or heterozygous novel germline mutations were validated by using Sanger sequencing (Fig 1,A). Widely altered C/EBPεPPIs To understand the functional effects of the p.Arg219His mutation, we assayed PPIs of WT and mutant C/EBPεusing J ALLERGY CLIN IMMUNOL NOVEMBER 2019 1366 G € O € OS ET AL Arg219 Arg219 His219 His219 His219 His219 Arg219 Arg219 bZIP Arg219His MGAP KDM6A CHD8 KDM1A ASH2L RBBP5 MSH6 PCF11 NFIA CPSF7 NFIB KIF4A KIF23 CHD7 CSK22 BCOR RCOR1 ARI3A FBRS DCAF7 QSER1 RCOR3 ARI3B SPDLY NIPBL CO039 NCOA3 PSPC1 TCF20 TF7L2 SRCAP MCAF1 MEF2D LDB1 NCOR2 TBL1X TRI33 HDAC3 SP130 TBL1R P66A SENP6 PIAS1 RFA1 PIAS2 LIN9 SAE2 LIN54 REQU T2FA TF3C4 GTF2I SNF5 ARI1B DPF1 SMRD2 SMRC2 RPRD2 SMCE1 SMRD1 ARI1A SMCA4 DIDO1 NACC1 ZHX3 ZBTB9 MTA3 BRD4 CICGPKOW ELF2 GSE1 FUBP2 BCL9 EMSA1 POGZ ZMYM4 ZN318 ZN638 RAI1 NCOA6 NCOA1 ZN644 TRPS1 ZMYM2 ZN608 ZN148 TIF1A CEBPE ZHX2 FOXP1 ZN609 FOXK1 ZN281 WIZ FOXC1 SATB2 CUX1 HOMEZ HXA10 ADNP SIX4 SATB1 EP400 DMAP1 EHMT2 EHMT1 CBP KMT2D PAXI1 TLE4 TLE1 HMGB1 ARNT UBP7 I2BP2 I2BP1 YLPM1 RBM14 RBM27 PRC2B FUBP1 SUGP1 RBM33 SLU7 PRCC CATIN DGC14 MINT WBP11 PPIL2 GPTC1 PRR12 SNW1 MAML1 EP300 CCNT1 Zinc finger proteins nBAF Homeobox Forkhead box DNA binding Histone methyltransferase RNA binding Notch enhancer complex U2-type spliceosome SUMO NCOA MLL1-WDR5 NCOR2/1ATN1 RNA polymerase RRM Lysine demethylase ARID RCOR RAVR1 ATP binding CF IIAm complex SMCA2 NCOR1 TF binding LINC CTF/NF-I family TLE IRF2BP PRR TCF Histone acetyltransferase TLE3 NCOA2 -3 -2 -1 0 1 SRCAP P66A SATB2 ARNT HMGB1 TRI33 LIN9 FOXK2 GTF2I NIPBL SATB1 CUX1 HOMEZ ZN148 EHMT1 ZHX3 SNW1 TIF1A ELF2 NCOA1 EHMT2 HXA10 MTA3 PIAS1 MCAF1 PIAS2 ZHX2 CEBPE interactin g p roteins log2 fold change (Arg219His / WT) positive regulation of transcription negative/positive regulation of transcription negative regulation of transcription A C B D E carrier affected I II III 12 131234567891011 12 2 14 34 5 6 7 8 910 1112 13 NC M/M M/M M/M M/+ +/+ M/+ +/+ M/+ M/+ +/+ +/+ +/+ M/+ M/+ M/+ IV 1 FOXK2 FIG 1. Pedigree of the index family and changes in PPIs. A, Pedigree of the index family with the C/EBPεArg219His mutation. Homozygous subjects are shown in red, heterozygous carriers are shown in orange, and deceased subjects are indicated by diagonal bars.B, Three-dimensional structure of a C/EBPεdimer (blue and red helixes) bound to a DNA fragment. The top right panel shows the WT Arg219-DNA interaction, and the right bottom panel shows the mutated 219His interaction. C, Schematic illustration showing the Arg219His mutation within the basic zipper (bZIP) region of the DNA-binding domain of C/EBPε.D, C/EBPεPPIs detected by using proximity-dependent biotin identification coupled to mass spectrometry. Interacting proteins were classified by using the CORUM and UniProt databases, and the complex and/or functional group membership are depicted by different colors. Novel interactions are shown with red edges, and the 2 previously known C/EBPεinteractions are shown in blue. E, The Arg219His mutation in C/EBPεcauses decreased associations with transcriptional repressors. Log 2 FCs of PPIs (mean, n 54) between Arg219His and WT C/EBPεare shown. Color coding highlights the transcription regulation action (UniProt) of proteins with more than 2-fold decrease in interaction (log 2 FC < 21). J ALLERGY CLIN IMMUNOL VOLUME 144, NUMBER 5 G € O € OS ET AL 1367 proximity-dependent biotin identification coupled to mass spectrometry (see Fig E2,A, in this article’s Online Repository at www.jacionline.org). This identified 144 C/EBPεinteraction partners, 141 of which were previously not reported (Fig 1,D, and see Table E1 in this article’s Online Repository at www.jacionline.org). Based on quantitative interaction analysis, 108 PPIs were significantly (P< .05) altered; 106 showed decreased and 2 showed increased interaction with mutant compared with WT C/EBPε(Fig 1,E, and see Table E1). Importantly, many of the diminished interactors were transcriptional repressors, suggesting widely dysregulated C/EBPε-driven transcription (Fig 1,E, and see Table E1). Twenty-four of the novel interactions were also seen in Jurkat T cells (see Table E1). We observed similar loss of transcriptional repressors when analyzing the mouse Arg219His C/EBPεmutant, suggesting a high degree of conservation of C/EBPεfunctions and effects of the mutation (Fig E2,B). 15 One of the known interactions was C/EBPεinteraction to SWI/SNF (SWItch/Sucrose Non-Fermentable) chromatin remodeling complex subunit SMARCC2 (SMRC2), which is an important regulator of myeloid differentiation. 23 This affinity was reduced in mutant compared with WT SMRC2 (log 2 Mut/WT 520.41). In addition to SMRC2, we found C/ EBPεto interact with 4 other SWI/SNF-related matrixassociated actin-dependent regulator of chromatin subfamily members(SNF5,SMRD1,SMRD2,andSMCE1),allofwhich were downregulated in patients compared with control subjects (see Table E1). To see how specific the changes in PPIs were for the Arg219His-mutated C/EBPε, we generated a cell line expressing Val218Ala C/EBPε, which is known to cause SGD, 14,24 and compared the interaction changes between these 2 mutants (see Fig E2,C, and Table E1). Most of the studied interactions did not differ between Val218Ala and WT C/EBPε. Increased chromatin occupancy of Arg219His C/ EBPε Mapping of the p.Arg219His mutation to the DNA-binding domain of C/EBPεprompted us to profile the chromatin occupancy of C/EBPε. We performed ChIP-seq from granulocytes without and with LPS stimulation (Fig 2 and see Table E2 in this article’s Online Repository at www.jacionline.org). Peak calling and overlap analysis from biological replicates revealed 3391 C/EBPε-binding sites in control subjects, 4686 in Arg219His heterozygote carriers, and 10322 in homozygous patients (Fig 2,A). Similar results were observed on LPS stimulation (Fig 2,B). Increased occupancy was also evident in TABLE I. Clinical characteristic of patients Patient II.2 II.7 II.13 Age at onset of symptoms 17 y 17 y Youth Current age Deceased at age 78 y 74 y 69 y Main symptoms Abdominal pain, high fever Crater-like ulcers of buccal mucosa occasionally during periodic fever Abdominal pain, high fever Abdominal pain, high fever Crater-like ulcers of buccal mucosa occasionally during periodic fever Duration of attacks (average) 4-5 d 4-5 d 4-5 d Frequency of attacks (average) Every 2-4 wk, later more seldom Every 2-4 wk, later more seldom Every 2-4 wk, later more seldom Other symptoms during attacks Lymphangitis Vomiting Myalgia Pleurisy Arthralgia Ileitis diagnosed by laparotomy Lymphangitis Pleurisy Episcleritis Scleritis Aphthous colitis Lymphangitis Vomiting Myalgia ESR (mm/h) at attacks 70-80 >50 70-80 Other major clinical events Myocardial infarction at age 46 y Operated on for ileal leiomyosarcoma at age 35 y Pyoderma gangrenosum at age 43 y Mesenterial lymph node biopsy at laparotomy at age 55 y showed granulomatous inflammation Recurrent respiratory tract infections since age 42 y Laparotomy at age 55 y showed ileitis and mesenterial lymph node biopsy granulomatous inflammation Infections Severe recurrent tongue abscesses when very young Easily acquired purulent wounds with delayed healing Paronychia Gluteal and submandibular abscesses Bleeding diathesis Moderately severe nose bleeds, as well as need for prolonged compression after needle sticks Postoperative hematomas In addition, patient II.3 was likely a carrier with similar systemic symptoms, periodic fever, and skin and mucosal sequelae. At age 18 years, she acquired rheumatic fever, causing mitral and aortic valve insufficiency and cardiac arrhythmias. At age 23 years, she had subacute endocarditis caused by Streptococcus viridans and died at age 34 years of ventricular fibrillation. Postmortem autopsy showed ‘‘substantial’’ numbers of calcified mesenteric lymph nodes with ‘‘nonspecific inflammation.’’ ESR, Erythrocyte sedimentation rate. J ALLERGY CLIN IMMUNOL NOVEMBER 2019 1368 G € O € OS ET AL C=3391 Ca=4686 P=10322 C=7149 Ca=13895 P=12849 CCa C LPS Ca LPS P LPS P AB Common to ca and p Common to all Unique to ca Unique to p Common to ca and p Common to all −1000 −500 0 500 1000 0 5 10 15 20 25 30 C Ca P CD WT Arg219His Relative distance from center (bp) Average profile Position Position Information content Information content 12345678910 0 0.5 1 1.5 2 0 0.5 1 1.5 2 12345678910 11 12 13 q q q q q p p Unique to p -5 kB +5 kB -5 kB +5 kB 1448 5618 2940 3290 2338 6339 3848 FIG 2. ChIP-seq analysis of C/EBPεDNA binding. Aand B, Area-proportional Venn diagrams of C/EBPεChIPseq binding sites and tag density maps of C/EBPε-binding events flanking 65 kb in the absence (Fig 2, A) and presence (Fig 2, B) of LPS treatment. C, Average C/EBPεChIP-seq signal profiles. D, Binding motif, as determined by using ChIP-seq. No significant changes were seen in the binding site of the Arg219His mutant. All ChIP-seq experiments were carried out in freshly isolated human granulocytes. C, Control subjects; Ca, heterozygous carriers; P, homozygous patients. J ALLERGY CLIN IMMUNOL VOLUME 144, NUMBER 5 G € O € OS ET AL 1369 average ChIP-seq signal intensities (Fig 2,C). Interestingly, de novo motif analyses identified highly similar consensus DNA-binding sequences in WT and mutant C/EBPε(Fig 2,D). This suggests that the increased C/EBPεchromatin occupancy was caused by mechanisms other than the altered DNA-binding motifs usually seen in neomorphisms. 25 FIG 3. Transcriptomic analysis of unstimulated (A-C) and bacterial LPSand DNA-stimulated (D-F) granulocytes using RNA-seq. Fig 3, A, RNA-seq revealed 464 differentially transcribed genes (FC > 2 and FDR < 0.05) between the patients and control subjects in unstimulated granulocytes. Of these, 198 (Fig 3, C) were identified to be interferon related by using Interferome (version 2.01, www.interferome.org), and 271 (Fig 3, D) had C/EBPε-binding sites (mapping to nearby genes within 650 kb). Importantly, 80 of these 271 genes were associated with patient-specific C/EBPεbinding. Fig 3, D, Similarly, 470 genes were differentially transcribed in bacterial LPSand DNAstimulated granulocytes. Fig 3, Eand F, Of these, 183 (Fig 3, E) were identified to be interferon related, and 289 (Fig 3, F) had C/EBPεbinding-sites. Eighty-one genes had patient-specific C/ EBPεbinding. Volcano plots represent log 2 FCs and 2log 10 FDRs of transcripts between the patients and control subjects. Different groups are color coded. C, Control subjects; Ca, heterozygous carriers; P, homozygous patients. J ALLERGY CLIN IMMUNOL NOVEMBER 2019 1370 G € O € OS ET AL Pronounced transcriptional changes in mutated unstimulated granulocytes Increased mutant C/EBPεbinding to DNA and decreased association with transcription repressors can lead to dysregulated transcriptome. Thus we compared transcriptomes of patients with CAIN and control subjects in unstimulated granulocytes using RNA-seq (Fig 3,A). This identified 464 significantly differentially transcribed (fold change [FC] >_ 2or<_ 0.5, false discovery rate [FDR] <_ 0.05) genes, 198 of which, including NLRP3 (FC 58.25), were interferon related (Fig 3,B, and see Table E3 in this article’s Online Repository at www.jacionline.org). Furthermore, Gene Ontology analysis revealed upregulation of genes involved in inflammatory responses, transcription, chemotaxis, and LPS response (see Table E4 in this article’s Online Repository at www.jacionline.org). One of the upregulated genes was PRTN3, which encodes the ubiquitous, especially in neutrophils, serine protease proteinase 3. 26 Activated neutrophils secrete PR3, which, among its other functions, cleaves structural proteins and activates the inflammasomeregulated cytokines IL-1band IL-18. 26,27 Upregulation of PRTN3 was verified by using RT-PCR (see Fig E3,A,inthis article’s Online Repository at www.jacionline.org). CEBPE was not among the differentially expressed genes in granulocytes; even slightly increased expression was detected by using RT-PCR in PBMCs (see Fig E3,B). To investigate whether these differentially transcribed genes were under C/EBPεcontrol, we mapped ChIP-seq peaks to nearby genes within 650 kb range and performed overlap analysis. Of the 464 differentially expressed genes, 271 had C/EBPε-binding sites, and importantly, 80 genes had patientspecific binding (Fig 3,C, and see Table E3). We noted increased occupancy and novel C/EBPε-binding sites in patients for overexpressed inflammasome-interleukin–related genes (eg, NLRP3 [see Fig E4,A, in this article’s Online Repository at www.jacionline.org], NFKBIA, and IL1R2), suggesting aberrant inflammasome activation. Comparison of results for differentially expressed genes with those of other studies Recently, Serwas et al 24 performed a proteomics analysis of neutrophils from patients with SGD (C/EBPεmutation p.Val218Ala). They detected decreased expression of several granule proteins and increased expression of proteins linked to the nucleoskeleton and cytoskeleton, such as nesprin, vimentin, and lamin B2. Because our proteomic analysis was performed to identify changes on PPIs and not changes in the proteome, we compared the differentially expressed proteins from Serwas et al with our differentially expressed genes in RNA-seq experiments. In our data, opposite to proteins of patients with SGD, nesprin-2 (SYNE2) was detected with decreased expression in neutrophils from patients with CAIN (see Table E3). Vimentin was detected with a 2.2-fold increase in patients with CAIN, but because of an FDR of 0.09 it was filtered out from the differentially expressed genes (FDR cutoff 50.05). Lamin B2 was not differentially expressed in RNA-seq analysis, but we found FIG 4. Transcriptomic analysis of IFN-a2–stimulated (Aand B) and IFN-g–stimulated (Cand D) granulocytes by using RNA-seq. Fig 4, A, RNA-seq revealed 534 differentially transcribed genes (FC > 2 and FDR < 0.05) between patients and control subjects after IFN-a2 stimulation. Fig 4, B, Of these, 266 had C/EBPε-binding sites (mapping to nearby genes within 650 kb), and importantly, 83 of these were associated with patientspecific C/EBPεbinding. Fig 4, C, Similarly, 427 genes were differentially transcribed in IFN-g–stimulated granulocytes. Fig 4, D, Of these, 208 had C/EBPε-binding sites, with 54 being associated with patientspecific C/EBPεbinding. Volcano plots represent log 2 FCs and 2log 10 FDRs of transcripts between patients and control subjects. Different groups are color coded. C, Control subjects; Ca, heterozygous carriers; P, homozygous patients. J ALLERGY CLIN IMMUNOL VOLUME 144, NUMBER 5 G € O € OS ET AL 1371 A B D G F C E FIG 5. Transcriptomic analysis and characterization of changes in inflammasome activation. A, mRNA levels of PBMCs from patients and subjects analyzed by using Nanostring technology. Custom gene panel with selected inflammasome, interleukin, JAK/STAT, and NF-kB1 pathway–related genes were used in analysis, and average FCs of 3 technical replicates are presented. Statistically significant changes (Student ttest) J ALLERGY CLIN IMMUNOL NOVEMBER 2019 1372 G € O € OS ET AL