Virginie Deswaerte, Paul Nguyen, Alison West, et al.
Mechanism
Cancer Cells by an IL18-Mediated Inflammation-Independent Inflammasome Adaptor ASC Suppresses Apoptosis of Gastric
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Fromathe Norwegian Institute of Public Health, Oslo, Norway;bthe Department of In-ternal Medicine, Diakonhjemmet Hospital, Oslo, Norway;cthe Bevital AS, Bergen, Norway;dthe Department of Clinical Science, University of Bergen and the Labora-tory of Clinical Biochemistry, Haukeland University Hospital, Bergen, Norway;ethe Department of Immunology, Rikshospitalet, Oslo University Hospital, Oslo, Norway;
fthe Department of Medical Biochemistry, Oslo University Hospital and the Institute of Clinical Medicine, University of Oslo, Oslo, Norway; andgthe Department of Pe-diatrics, Østfold Hospital Trust, Gralum, Norway. E-mail:karlmarild@gmail.com.
The Norwegian Mother and Child Cohort Study (MoBa) was supported by the Norwe-gian Ministry of Health and Care Services and the Ministry of Education and Research, National Institutes of Health (NIH)/National Institute of Environmental Health Sciences (NIEHS) (contract no. N01-ES-75558), NIH/National Institute of Neurological Disorders and Stroke (NINDS) (grant no. 1 UO1 NS 047537-01 and grant no. 2 UO1 NS 047537-06A1). This subproject within MoBa is supported by the Norwegian Research Council (grant no. 221909/F20). The genotyping service for this study was financed by Helse Sør-Øst. K.S. was funded through an unrestricted grant from Oak Foundation, Geneva, Switzerland. The funding sources did not influ-ence the design, conduct, and reporting of the present study.
Disclosure of potential conflict of interest: O. Midttun is an employee at Bevital, which carried out the analyses on cord blood. P. M. Ueland is a member of the steering board of the nonprofit Foundation to Promote Research into Functional Vitamin B12Deficiency, which owns Bevital. J. P. Berg receives payment for lectures from MSD and Boehringer Ingelheim. L. C. Stene receives grant support from the Research Council of Norway. K. Størdal receives grant support from Oak Founda-tion, Geneva. The rest of the authors declare that they have no relevant conflicts of interest.
REFERENCES
1.Green PH, Lebwohl B, Greywoode R. Celiac disease. J Allergy Clin Immunol 2015;135:1099-106; quiz 107.
2.Lindehammer SR, Bj€orck S, Lynch K, Brundin C, Marsal K, Agardh D, et al. Early human pregnancy serum cytokine levels predict autoimmunity in offspring. Auto-immunity 2011;44:445-52.
3.Marild K, Ludvigsson JF, Størdal K. Current evidence on whether perinatal risk factors influence coeliac disease is circumstantial. Acta Paediatr 2016;105:366-75.
4.Magnus P, Birke C, Vejrup K, Haugan A, Alsaker E, Daltveit AK, et al. Cohort profile update: the Norwegian Mother and Child Cohort Study (MoBa). Int J Epi-demiol 2016;45:382-8.
5.Midttun O, Hustad S, Ueland PM. Quantitative profiling of biomarkers related to B-vitamin status, tryptophan metabolism and inflammation in human plasma by liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom 2009;23:1371-9.
6.Radunovic N, Kuczynski E, Rebarber A, Nastic D, Lockwood CJ. Neopterin con-centrations in fetal and maternal blood: a marker of cell-mediated immune activa-tion. Am J Obstet Gynecol 1999;181:170-3.
7.Trynka G, Hunt KA, Bockett NA, Romanos J, Mistry V, Szperl A, et al. Dense gen-otyping identifies and localizes multiple common and rare variant association sig-nals in celiac disease. Nat Genet 2011;43:1193-201.
8.Kvalvik LG, Nilsen RM, Skjaerven R, Vollset SE, Midttun O, Ueland PM, et al.
Self-reported smoking status and plasma cotinine concentrations among pregnant women in the Norwegian Mother and Child Cohort Study. Pediatr Res 2012;72:
101-7.
9.Skevaki C, Van den Berg J, Jones N, Garssen J, Vuillermin P, Levin M, et al. Im-mune biomarkers in the spectrum of childhood noncommunicable diseases.
J Allergy Clin Immunol 2016;137:1302-16.
Available online November 16, 2016.
http://dx.doi.org/10.1016/j.jaci.2016.10.016
Life-threatening NLRC4-associated hyperinflammation successfully treated with IL-18 inhibition
To the Editor:
Clinical application of several rapidly evolving technologies
—next-generation DNA sequencing, biomarker discovery, and targeted cytokine blockade—has been particularly beneficial to understanding an expanding spectrum of genetically defined autoinflammatory diseases.1Our understanding of the pathways that cause hemophagocytic disorders, such as macrophage
activation syndrome (MAS) and hemophagocytic lymphohistio-cytosis (HLH), is evolving similarly. MAS and HLH are life-threatening sepsis-like conditions notable for hyperferritinemia, acute cytopenias, and hepatitis. If not promptly recognized and treated, they can progress to consumptive coagulopathy, hemophagocytosis, multiorgan failure, and high mortality. HLH is classically associated with genetic defects in cytotoxicity, whereas MAS is observed as a complication of rheumatic diseases.1
We recently implicated gain-of-function mutations in NLRC4, a protein that activates the inflammasome, in a syndrome of recurrent MAS with early-onset enterocolitis (NLRC4-MAS, OMIM#616050).2,3 Inflammasomes are large innate immune complexes that quickly and exponentially catalyze the activation of pro–IL-1b and pro–IL-18. Although IL-1b blockade is effective in many ‘‘inflammasomopathies,’’1,2 the role of IL-1b in MAS is controversial. IL-1 blockade is effective in treating MAS-prone diseases, but was not protective against the development of MAS. The effects of blocking IL-18 are unknown. Patients with NLRC4-MAS have extraordinary and chronic elevation of serum IL-182,3; and although extraordinary IL-18 levels are a feature of MAS more generally, IL-18 is only modestly elevated in other genetic inflammasomopathies.
Classically, myeloid cell–derived IL-18 enhances IL-12–
driven IFN-gproduction, but IL-18 can also synergize to promote various diverse immunological effects. Notably, IFN-g is the cytokine most implicated in driving familial forms of HLH,4 although its role in MAS is more controversial. IL-18 is also highly expressed in intestinal (and other) epithelial cells and has complex roles in intestinal homeostasis and colitis.5IL-18 binding protein (IL-18BP) is an endogenous protein that binds tightly to IL-18, preventing signaling but not serologic detection.6
Herein, we report a previously healthy white female who developed a parainfluenza upper respiratory tract infection at age 6 weeks. Cough, coryza, and viral RNA cleared, but hectic fevers and erythrodermic rash (Fig 1,Aand D) persisted for several weeks. After 3 weeks of fever, she acutely developed oral intolerance and severe secretory diarrhea that persisted despite cessation of oral intake. Her condition worsened with acute rise in inflammatory markers, relative thrombocytopenia, and rising ferritin, consistent with an MAS-like syndrome. Thorough infection and malignancy workups were unrevealing, and there were no signs of immunodeficiency. Endoscopy showed severe mucosal ulcerations and inflammation extending from stomach through large intestine, with normal staining for intestinal regulatory T (Treg) cells (Fig 1,BandC; seeFig E1,A, in this article’s Online Repository at www.jacionline.org). Functional assessment of cytotoxicity was normal. Clinical whole-exome sequencing returned a de novo heterozygous mutation in NLRC4(c.1022T>C, p.Val341Ala) within 2 weeks. This same mutation had previously been associated with a very similar, but dominantly inherited syndrome in a small kindred, including an infant who succumbed soon after birth.3
The patient was treated aggressively with corticosteroids (including 11 pulses of 30 mg/kg in 1 month) and IL-1 blockade (10 mg/kg/d anakinra) with minimal response (Fig 1,D). The addition of maximal doses of TNF-a-blockade (infliximab, up to 20 mg/kg), cyclosporine, and a4b7-integrin inhibition (vedolizumab) resolved her fever and coagulopathy but did not
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improve anemia, hyperferritinemia, or enterocolitis. We assessed serum for total IL-18, as well as free IL-18 using a novel assay,6 and found both to be extraordinarily elevated (Fig 2,A). Under an emergency compassionate-use Investigational New Drug authorization from the Food and Drug Administration, the patient was given recombinant human IL-18BP (rhIL-18BP) at a dose of 2 mg/kg subcutaneously every 48 hours. Within the first 2 doses, the patient’s overall demeanor improved rapidly in correlation with an acute drop in ferritin (Fig 1,D). C-reactive protein and enterocolitis also improved, and the patient successfully restarted enteral feeding 11 days after rhIL-18BP initiation. While on combined IL-18 and IL-1bblockade, the patient also weaned from all other forms of immunosuppression (Fig 1,D). Given the severe presentation, persistent total IL-18 elevation, and chronic course associated with this mutation,3 further attempts at weaning were gradual. The patient remains well after 11 months of combined IL-1b and IL-18 blockade, having weathered vaccination (except live-virus vaccines), typical infections, and partial weaning of IL-1bblockade without flare.
To better understand the pathogenic mechanisms at work, we measured IL-18 and related cytokines in serum and stool, and obtained whole blood transcriptional profiles. Serum analysis demonstrated massive, chronic, and stable serum total IL-18 elevation, consistent with our original reports (Fig 2,A).2,3Serum levels of CXCL9, a chemokine induced by IFN-g (and other inflammatory mediators) and associated with HLH activity,7 remained high despite high-dose steroids and IL-1 inhibition, but fell after initiation of rhIL-18BP (Figs 1 and 2, A). Free
IL-18 fell slightly with immunosuppression, but quickly fell to normal with rhIL-18BP. In stool, we measured almost 15-fold less total IL-18 than in serum, but minimal IL-18BP, resulting in nearly 3 times more free IL-18 (Fig 2,B). rhIL-18BP treatment correlated with a brief spike in stool IL-18BP levels. All cytokines decreased with clinical improvement of gut disease.
Whole blood transcriptional analysis from flare and after clinical improvement was compared with similar profiles in a previously described patient with NLRC4-MAS,2 and several patients with neonatal-onset multisystem inflammatory disease (NOMID, OMIM 607115) withNLRP3 mutations. Transcripts encoding neutrophil-associated genes (CD177, HP), components of the IL-18 receptor (IL18RAP),NLRC4itself, andNAIP(known to promote NLRC4 inflammasome assembly) were elevated during flare in both NLRC4-MAS and NOMID samples (Fig 2, C). Elevated levels ofNLRC4transcripts have also been observed in patients with active systemic juvenile idiopathic arthritis (sJIA).8However, there were prominent decreases in transcripts associated with cytotoxic T and natural killer cells (EOMES, TBX21, CD8A, PRF1), as well as Treg cells (FOXP3, CTLA4), during disease flare most pronounced in patients with NLRC4-MAS (Fig 2,C). Network analysis of genes upregulated or down-regulated in NLRC4-MAS identified an IFN-g–associated network as the most enriched (Fig 2,D; seeTable E1in this arti-cle’s Online Repository atwww.jacionline.org). Taken together, these transcriptional findings support myeloid cell activation and cytotoxic and regulatory T-cell dysfunction as potential pathomechanisms.
FIG 1. Rash, enterocolitis, and clinical course in NLRC4-related autoinflammation:A,Erythrodermal skin lesions.B,Ulcerative duodenal plaques.C,Edematous sigmoid colon with flattened villi, crypt drop-out, and apoptotic crypt epithelial cells(inset circles); 203, H&E.D,C-reactive protein (CRP), ferritin, enteral intake, and anti-inflammatory medications. Red box: MAS onset. Red line: start of rhIL-18bp. H&E, Hematoxylin and eosin. *Steroid(gray outline): running 7-day average of daily prednisone-equivalent dose.
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Rational choice of immunomodulatory therapies in monogenic inflammatory diseases can be challenging. Although our recent reports reinforced IL-18 as an enticing target for this patient, our previous measurements suggested possibly more IL-18 than could be reasonably neutralized (often more than 100,000 pg/mL).
However, the free 18 assay demonstrated levels of unbound IL-18 consistent with the potential for pharmacologic inhibition,6and enabled us to verify free IL-18 normalization soon after treatment initiation (Fig 2,A). Importantly, rhIL-18BP was administered with and after multiple other immunosuppressive drugs (Fig 1,D). Its beneficial effects may have been mediated by improving other drugs’ efficacy or pharmacodynamics (intestinal protein losses, in particular).
Interestingly, IL-18 remains chronically elevated long after patients with NLRC4-MAS have clinically improved2(Fig 2,A),
demonstrating that IL-18 alone is not sufficient to drive MAS.
Likewise, natural killer cells from patients with sJIA may become chronically insensitive to IL-18.9 The rapidity of our patient’s response to rhIL-18BP suggests inhibition of ongoing IL-18 signaling rather than release of IL-18 insensitivity. Her durability of response suggests disruption of one of the amplification loops (possibly involving IFN-g) that often underlie autoinflam-matory diseases.1 The chronicity of serum (but not blood transcriptional) IL-18 elevation in NLRC4-MAS suggests a possible nonhematopoietic source, and measurements in stool suggest minimal baseline luminal secretion and complex tissue-specific regulation.
Gene expression analysis identified potentially generalizable patterns of myeloid cell activation and Treg-cell suppression shared between NLRC4-MAS and NOMID, but also NLRC4-specific
FIG 2.Serum, stool, and transcriptional analyses: Serum(A)and stool(B)cytokines (in pg/mL) and serum ferritin (gray, ng/mL). Upper limit of healthy controls, if visible, crosses the y-axis.CandD,Whole blood RNA-seq comparing flare versus posttreatment in this patient (V341A), another patient with NLRC4-MAS (T337S),2and 7 patients with NOMID. Log10fold-change for genes higher (red) or lower (blue) during flare in both patients with NLRC4-MAS. See alsoTable E1.D, Most-enriched transcriptional network generated by the differentially expressed gene list. *Upper limit of detection.
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1700 LETTERS TO THE EDITOR
depression of cytotoxicity-related transcripts reminiscent of HLH.
Importantly, these transcriptional findings could represent shifts in cellular composition caused by steroid use or acute inflammation rather than underlying pathophysiology.
This report raises hope for more general benefit treating diseases marked by excessive free IL-18 and also preventing progression to MAS in susceptible patients. After nearly a year of chronic IL-18 blockade, there are no signs of significant immunosuppression or dysregulated mucosal homeostasis (normal growth, and lung and intestinal function). In conclusion, this patient’s life-threatening autoinflammatory condition improved because of rapid clinical and genetic diagnosis, identification of a novel biomarker, and suc-cessful deployment of an experimental, targeted therapy. The patient’s improvement, coupled with ongoing studies in HLH and sepsis, help define relevant roles for IL-18, IL-1b, and IFN-gin sepsis-like systemic inflammation. Raphaela Goldbach-Mansky, MD, MHSaà Edward M. Behrens, MDc FromaIntramural Research Program, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, Md;bthe Division of Rheumatology, Department of In-ternal Medicine Specialties, University Hospitals of Geneva, Geneva, Switzerland;
cthe Children’s Hospital of Philadelphia, Philadelphia, Pa; anddAB2Bio, Ltd, Lau-sanne, Switzerland. E-mail:scott.canna@chp.edu.
*Scott W. Canna, MD, is currently affiliated with Children’s Hospital of Pittsburgh, Pittsburgh, Pa.
àAdriana de Jesus, MD, PhD, and Raphaela Goldbach-Mansky, MD, MHS, are currently affiliated with IRP National Institute of Allergy and Infectious Diseases, Bethesda, Md.
S.W.C., L.M., A.de.J., and R.G.-M. were supported by the Intramural Research Program of the National Institute of Arthritis, Musculoskeletal, and Skin Diseases. E.M.B. was supported by the National Institutes of Health (grant no. R01 HL112836-A1), the Nancy Taylor Foundation, and Sean Fischel Connect. C.G. is supported by Swiss Na-tional Science Foundation (grant no. 10030_152638), the Rheumasearch Foundation, and the Institute of Arthritis Research.
Disclosure of potential conflict of interest: C. Girard’s institution has received a grant from AB2Bio. J. Kelsen’s institution has received grant K23 from the National Insti-tutes of Health (NIH). A. Sleight and E. Schiffrin are employed by AB2 Bio, Ltd. C.
Gabay has received a grant and consulting fees from AB2 Bio for this work and con-sultancy fees from Roche, BMS, Merck, Pfizer, Novartis, Celgene, and Sanofi for other work. R. Goldbach-Mansky’s institution has received grants from SOBI, Lilly, Regeneron, and Novartis. E. M. Behrens’ institution has received a grant from the NIH, Sean Fischel Connect, and Nancy Taylor Foundation; he has received a consul-ting fee from AB2Bio and a grant from the NIH. The rest of the authors declare that they have no relevant conflicts of interest.
REFERENCES
1.de Jesus AA, Canna SW, Liu Y, Goldbach-Mansky R. Molecular mechanisms in genetically defined autoinflammatory diseases: disorders of amplified danger signaling. Annu Rev Immunol 2015;33:823-74.
2.Canna SW, de Jesus AA, Gouni S, Brooks SR, Marrero B, Liu Y, et al.
An activating NLRC4 inflammasome mutation causes autoinflammation with recurrent macrophage activation syndrome. Nat Genet 2014;46:1140-6.
3.Romberg N, Al Moussawi K, Nelson-Williams C, Stiegler AL, Loring E, Choi M, et al. Mutation of NLRC4 causes a syndrome of enterocolitis and autoinflamma-tion. Nat Genet 2014;46:1135-9.
4.Jordan MB, Hildeman D, Kappler J, Marrack P. An animal model of hemophagocytic lymphohistiocytosis (HLH): CD81T cells and interferon gamma are essential for the disorder. Blood 2004;104:735-43.
5.Nowarski R, Jackson R, Gagliani N, de Zoete MR, Palm NW, Bailis W, et al. Epithe-lial IL-18 equilibrium controls barrier function in colitis. Cell 2015;163:1444-56.
6.Novick D, Kim SH, Fantuzzi G, Reznikov LL, Dinarello CA, Rubinstein M. Inter-leukin-18 binding protein: a novel modulator of the Th1 cytokine response. Immu-nity 1999;10:127-36.
7.Takada H, Takahata Y, Nomura A, Ohga S, Mizuno Y, Hara T. Increased serum levels of interferon-gamma-inducible protein 10 and monokine induced by gamma interferon in patients with haemophagocytic lymphohistiocytosis. Clin Exp Immunol 2003;133:448-53.
8.Quartier P, Allantaz F, Cimaz R, Pillet P, Messiaen C, Bardin C, et al. A multicentre, randomised, double-blind, placebo-controlled trial with the interleukin-1 receptor antagonist anakinra in patients with systemic-onset juvenile idiopathic arthritis (ANAJIS trial). Ann Rheum Dis 2011;70:
747-54.
9.de Jager W, Vastert SJ, Beekman JM, Wulffraat NM, Kuis W, Coffer PJ, et al.
Defective phosphorylation of interleukin-18 receptor beta causes impaired natural killer cell function in systemic-onset juvenile idiopathic arthritis. Arthritis Rheum 2009;60:2782-93.
Available online November 19, 2016.
http://dx.doi.org/10.1016/j.jaci.2016.10.022
Rapamycin preferentially inhibits human IL-51TH2-cell proliferation via an mTORC1/S6
kinase-1–dependent pathway
To the Editor:
The mechanistic target of rapamycin (mTOR) is a serine/
threonine kinase that integrates metabolic and activation signals to regulate cell proliferation and differentiation. mTOR plays a major role in T-cell proliferation and differentiation.1-3 The transplant drug rapamycin is the canonical inhibitor of mTOR and in the 5 to 10 nM concentration range used clinically, rapamycin largely exerts its effect via inhibition of mTOR complex 1 (mTORC1).4
Recently, several groups have characterized an IL-51 pathogenic effector TH2 (peTh2) subpopulation with enhanced effector function.4 Notably, peTH2 cells demonstrate greater proinflammatory function in murine models of asthma and atopic dermatitis (AD) and are increased in human eosinophilic gastrointestinal diseases (EGID) and AD.5-9 Because of their pathogenic role in eosinophilic inflammation, we sought to examine mTOR function within the human peTH2 subpopulation in samples from subjects with eosinophilic asthma or EGID (see Table E1in this article’s Online Repository atwww.jacionline.
org).
To investigate the mTOR pathway in human TH1 and TH2 cell responses, we used dye dilution proliferation cultures coupled with intracellular cytokine staining (see this article’s Methods section in the Online Repository at www.jacionline.org) to examine the dominant TH1- or TH2-cell proliferative responses associated with respiratory syncytial virus or house dust mite (HDM) antigen, respectively (seeFig E1in this article’s Online Repository at www.jacionline.org). Although rapamycin inhibited the output of both TH1 and TH2 cells in these proliferation cultures, the greatest inhibition was seen in IL-5–expressing cells (Fig 1,A). Accordingly, a dose response using a Boolean gating strategy was performed to examine the differential effect of rapamycin on IL-51TH2 (IL-51, IL-131) versus IL-52TH2 (IL-52, IL-131) versus TH1 (IFN-g1) cells.
J ALLERGY CLIN IMMUNOL VOLUME 139, NUMBER 5
LETTERS TO THE EDITOR 1701