Accepted Article Institute Infectious Diseases, National 20892 Maryland, Maryland, 21702 Cancer LaboratoryFrederick, for Research, National Frederick Inc., Research, Leidos Biomedical Directorate, Bethesda, Maryland, Health, 20892 of Institutes This by is protectedarticle reserved. Allrights copyright. doi: 10.1111/all.13146 lead to differences between this version and the throughbeen the copyediting, pagination typesetting, which process, may and proofreading hasThis article been accepted publicationfor andundergone peerfull but review not has 4 3 2 1 PhD Prakash Babu, Senbagavalli eosinophilia familial in 5expression of interleukin Dysregulation Article type : Articl Original 16-Feb-2017 : Date Accepted Revised Date : 15-Feb-2017 Received : Date 18-Nov-2016 DR. AMYKLION (Orcid ID : 0000-0002-4986-5326) MSc Genomic Technologies Section, Research Technologies Branch, National Institute of Allergy and and Allergy of Institute National Branch, Technologies Research Section, Technologies Genomic Bethesda, Health, Institutes of National Medicine, Center, LaboratoryDepartment Clinical of Research and Developmental Applied Laboratory Human and Immunoinformatics, of Retrovirology National and Infectious Diseases, of Allergy Institute National Diseases, Parasitic Laboratory of 2 , Irina Maric, MD 3 ,Timothy G. Myers, G.Myers, PhD ,Timothy e: Experimental Allergy e: and 1 , Yun-Yun K. Chen, MD Chen, K. Yun-Yun , s of Health, Bethesda, Maryland, 20892 20892 Maryland, Bethesda, s Health, of 4 , Thomas B. Nutman, MD B. Nutman, , Thomas Version of Record. Please Version as this cite ofRecord. article 1,* , Sandra Bonne-Annee, PhD Bonne-Annee, Sandra , Immunology 1 , Amy D. Klion, MD Amy Klion, , D. 1 , Jun Yang, Yang, , Jun 1 . Accepted Article This by is protectedarticle reserved. Allrights copyright. No.HHSN261200800001E. under Contract Health, of Institutes National Institute, Cancer from theNational funds federal or in with whole beenpart in funded has project This NIH. NIAID, Research, Intramural of Division bythe wasfunded The work Funding: [email protected] Email: 301-451-2029 Fax: 301-435-8903 Telephone: Bethesda, MD 20892 4 B1-28, Drive 4, Memorial Room Building Health of Institutes National Diseases, of and AllergyInfectious Institute National Parasitic Diseases, of Laboratory M.D. Amy Klion, D. Author: Corresponding MD 21215 Baltimore, of Hospital Sinai Baltimore Medicine of Department M.D. Chen, Kathy Yun-Yun *Present affiliation: Accepted Article This by is protectedarticle reserved. Allrights copyright. stimulated both PBMC and from cultures. ILC2 levels supernatants protein in IL-5 as affected family CD14+,were CD19+ andILC2members, CD3+ CD4+, from purified cells note, Of members. family affected increased in ( cytokines of other Th2 mRNA expression increased by accompanied was not this members, family affected Results immunoassays. multiplexed array by suspension assessed were supernatants culture and PBMC serum in levels Cytokine kindred. large asingle to belonging members family and unaffected subsets affected cell from purified and cells mononuclearin blood (PBMC), peripheral Methods. Objective. lineage. eosinophil the in abnormality a primary and studieshave demonstrate to prior failed FEthe of cause genetic isunknown, (>1500/ eosinophilia peripheral by lifelong of thepresence Background Abstract of haveinterest. conflict the a Noneof Interest: authors of Conflict declared paper. the of editing writing and the and the experiments analysisof and thedesign in participated Amy Klion and Nutman Thomas paper, the of writing the and data the analysis of the of experiments, execution and the designin participated Timothy and Myers Yang, Jun Bonne-Annee, Sandra Chen, K. Yun-Yun Babu, Prakash Senbagavalli Contributions: Author . Whereas . Whereas Microarray analysis and real-time PCR were used to examine transcriptional differences in in differences transcriptional examine to used were PCR real-time and analysis Microarray The aim of the present study was to identify the cells driving the eosinophilia in FE. study was in aim The toidentify present cellsthe eosinophilia the driving of IL4 . Familial . (FE) Familial eosinophilia is a rare disorder inherited dominant autosomal characterized or or IL5 IL13 mRNA expression was significantly increased in freshly isolated PBMC from from PBMC isolated freshly in increased significantly was expression mRNA ). Serum levels IL-5 of andreceptorIL-5 IL5 mRNA expression was significantly increased in increased significantly was mRNA expression μ L). Mapped to chromosome 5q31-q33, L). tochromosome 5q31-q33, Mapped α , but not IgE, similarly not were , but Accepted Article This by is protectedarticle reserved. Allrights copyright. members Unaffected family UA chain reaction polymerase Quantitative transcriptase reverse cells bloodmononuclear Peripheral qRT-PCR Odds Logarithm of PBMC LOD Hypereosinophilia HE Healthy HD controls Geometric mean GM Familial hypereosinophilic Familial eosinophilia syndrome FHES FE Cycle Ct threshold Affected A family Abbreviations members 5. interleukin cytokine; dominant; syndrome; autosomal hypereosinophilic Eosinophil; Words Key subsets). component their (and in PBMC IL-5 production of dysregulation Conclusions.

These data are consistent with the hypothesis that the eosinophilia in in FE to is secondary theeosinophilia hypothesisthe that with are These consistent data Accepted Article This by is protectedarticle reserved. Allrights copyright. in FE. eosinophilia the driving family members (1). The aim of the present study was to identify the cell population(s) responsible for seIL-5 IL-5relatively in was insensitive, detecting measure used to assay the family members, unaffected and between affected similar previously were measured levels cytokine serum Although (3). defect eosinophil primary a for evidence little provided have assays, survival eosinophil and cells, CD34+ from eosinophils of vivo differentiation ex morphologic including examination, Prior studies, thanthe eosinophil. byproduced other types cell tomediators be or lineage inthesubjectsFE with duea primary to abnormality could eosinophil to date. have bybut unsuccessful genome are whole mutation ongoing, been sequencing their promoters has failed toidentify thecausative including genes, candidate of sequencing targeted 6.49), LOD of score (multipoint cluster thecytokine gene onchromosome containing 5q toa one region large kindred (3). proteins granule eosinophil of and release markers activation surface morphology, cellular by asevidenced activation eosinophil eosinophilia are This (2,3). “benign” uncommon phenot affected Despite familyin prolonged eosinophilia >1500/ eosinophilia Introduction The increased levels of morphologically normal peripheral blood eosinophils observed in observed in eosinophils peripheral blood normal Themorphologically levels increased of FE widelinkageby analysis in for has beengenome mapped genethe Although responsible peripheral sustained of presence the by (FE) characterized is a eosinophilia Familial disorder μ L beginning at birth that has an autosomal dominant inheritance pattern (1,2). (1,2). pattern inheritance has dominant anautosomal Lthat at birth beginning members, clinical clinical relatedtothemembers,manifestations mutation (4). Attempts to identify the causative ra from only 3/14 affectedra from 3/22 unaffected and ype is likely related to a relative lack of lack arelative to related likely ype is IL5 , IL3 , and , and GMCSF , and Accepted Article (Invitrogen) at at -80 (Invitrogen) Trizol stored or in Fisher Scientific) Thermo (DMSO; sulfoxide %dimethyl Invitrogen) and 7.5 (FBS; serum bovine fetal 10% C–RPMIcontaining medium with freezing in nitrogen liquid in cryopreserved were PBMC of Aliquots blue. trypan with staining by determined were viability and Cell number CA). Carlsbad, (Invitrogen, buffer lysing (ACK) ammonium-chloride-potassium with 10minutes temperature at room lysed for were Red cells Sweden). Uppsala, PLUS, Healthcare, GE PBMC purification fields. overcount 10high-powered average as arethe reported Results tryptase immunostaining. following hematopathologist a single by biopsies core marrow bone in quantified were cells Mast Center. Clinical NIH Laboratory Medicine, of Department the in performed was cytometry, flow by blood T, Bof andNKcell whole in subsets levelsassessment and counts, serumblood immunoglobulin including complete testing, eosinophilia. Routine of laboratory manifestations and endorgan assess eosinophilia of causes secondary exclude to evaluation clinical extensive an underwent subjects (3), previously consent. As described informed gave written participants All research (NCT00001846). samplesfor blood normal to obtain protocol clinical board-approved review institutional ona separate were recruited without (HC) eosinophilia controls Healthy Figure Supplemental 1). and a protocol that eosinophilia (NCT00091871 prior generations review protocols on wereinstitutional tostudy evaluated clinical familial board-approved count absolute (HE; eosinophil >1500/ hypereosinophilia This by is protectedarticle reserved. Allrights copyright. Study subjects and methods population Patient PBMC were isolated from peripheral blood by density gradient centrifugation (Ficoll-Paque gradient bloodbycentrifugation density peripheral PBMCfrom isolated were dominant autosomal with twokindreds from members family Affected andunaffected

o C for future use. preceded clinicaltrials.gov) (see pedigrees in pedigrees (see clinicaltrials.gov) preceded μ L) documented over L) at leastthree documented over in vitro

Accepted Article This by is protectedarticle reserved. Allrights copyright. Kit PCRusing QIAQuick purification the and purified (Invitrogen) T4 DNApolymerase with cDNA was blunt-ended (Invitrogen). I, andDNA ligase DNA polymerase RNaseH, with E. coli was obtained cDNA synthesis IISecond-strand (Invitrogen). and Superscript a using dT primerT7 oligo prior to reverse-transcription (24) (Invitrogen) instructions manufacturer’s analysis Microarray Figure in Supplementaryprovided are The 2 strategies gating instructions. manufacturer’s Supplemental found Table 1. in used antibodies can be of A listing (5). complete asdescribed ILC3 (CRTH2- previously CD117+) and ILC2 (CRTH2+) CRTH2-), (CD117- andILC ILC1 subsets as as LIN-CD45+CD127+ defined were ILCs subsets. (ILC) cells innate lymphoid sort to markers subset with and cells positive lineage alineagedeplete to of cocktail with markers PBMC stained were monocytes. Fresh andCD14+ cells B ofT CD19+ and CD3+CD8+ CD3+CD4+ cells, isolation for CD14 and CD19 CD3, CD8, CD4, subsets of PBMC sorting Flow CD3- isolated. were T cells unlabeled the and CD3+ labeled T inan the magnetically separator, selection Pro cells autoMACS family members were magnetically labeled with CD3 Microbeads (Miltenyi Biotec). Using positive PBMC subsets CD3+ CD3- and of enrichment Magnetic Labeling was performed using ENZOthe Labeling was performed BioArray Total RNA was isolated from freshly purified PBMC stored in Trizol according to the tothe according Biosciences) (BD sorter II cell FACSAriaTM a using sorted were All cells to antibodies with stained were controls and healthy affectedPBMC family members from unaffected affected and from samples) cryopreserved from previously or fresh PBMC (either

TM HighYield TM TM RNA Transcript Kit (Affymetrix) (Affymetrix) Kit RNATranscript (Qiagen, Germantown, MD). MD). Germantown, (Qiagen, . Accepted Article (Hs00174379_m1), (Hs00174379_m1), This by is protectedarticle reserved. Allrights copyright. using Taqman qRT-PCR Biosystems). (Applied kit Transcription Reverse cDNA Capacity High to the manufacturer’s instructions (Invitrogen). Total RNA (1 PBMC of subsets and PBMC qRT-PCR analysisof Real-Time dataset. the arrays in all across each set probe for normalized were set. probe Values that of mRNA target the of expression estimate to set eachprobe for level signal adjusted an computes Gene Chip Operating System (GCOS) software using the MAS5 algorithm. The MAS5 algorithm (6) Affymetrix using analyzed were images Array 3000. Scanner GeneChip Affymetrix the using scanned stai followed byand hours 18 washing Genechips for U133Plus 2.0 onAffymetrix Affymetrix controls B2 and were from hybridized Oligo hybridization cRNA with Fragmented CA). Santa Clara, (Agilent, ona Bioanalyzer assessed was and quality the by OD, quantified cRNAlabeled was purification, After instructions. the manufacturer’s according to probes (Taqmanprobes Invitrogen, SanDiego, in24-well tissueCA) culture plates (Costar, Corning, NY). Afterresting from pyruvate sodium (all L-glutamine(10mM), HEPES (2mM), gentamicin, supplemented with medium, RPMI 1640 in andresuspended washed were thawed, PBMC use.Isolated future supernatants PBMC and serum in soluble mediators of Measurement the presence of sufficient cDNA (18S cDNAC (18S sufficient of the presence cycle threshold (C cycle threshold rRNA 18S corresponding the to normalized was expression relative the and triplicate, in performed Total RNA wasextracted from PBMC or from flow sorted PBMC subsets in Trizol according Serum was collected from whole blood by centrifugation and stored immediately at −80°C for at immediately for and by−80°C stored centrifugation blood whole from Serum wascollected R Fast Universal PCR Master Mix using commercially available FAM-MGB primer FAM-MGB primer available commercially Mix using PCR Master Universal Fast R Gene Expression Assay, Applied Biosystems) for for Biosystems) Applied Assay, Gene Expression t ). Lack of expression was defined as a as C defined was Lackexpression of ). IL4 (Hs00174122_m1) and (Hs00174122_m1) t <12). Data are expressed as 1/ expressed Data are <12). IFN- γ ning on Affymetrix Fluidic 400. The arrays were were The arrays 400. Fluidic Affymetrix ning on (Hs00174143_m1). All reactions were All reactions (Hs00174143_m1). t of more than 35 cycles (for (for cycles 35 than more of μ g) was reverse-transcribed using the using the g) wasreverse-transcribed IL5 Δ (Hs00174200_m1), (Hs00174200_m1), C t .

was carried out IL5 ) despite IL13

Accepted Article This by is protectedarticle reserved. Allrights copyright. 2x10 °C. -80 at stored and harvested were supernatants 37°C. at culture hours 18 The for respectively) ng/mL ng/ml, and 150 15 PMAwithout and concentrations: with (final and thecells cultured overnight, ionomycin were AB serum, 10 U/mL of IL-2 (PeproTech) and 50 ng/mL of rIL-7 (PeproTech) and activated with using the False Discovery Rate (FDR) method (8). (8). method Rate (FDR) using False the Discovery testing multiple for P-values adjusted Cary were Institute, NC). 8.0 (SAS version JMP/Genomics in ANOVA procedure theSAS mixed-effects using groups two the between differences expression before calculating normalization by quantile was transformed 8unaffected) affected subjects, 17 arrays for the MAS5 algorithm) (9 (using estimates data, thetable set expression gene probe of rank test.A p-value of <0.05 wasconsidered statis signed Wilcoxon the using analyzed were samples paired and test, Mann-Whitney the using compared analysis Statistical (7). described as ELISA capture previously using chemiluminescence an in-house (sIL-5R alpha receptor IL-5 Soluble instructions. manufacturer’s measured by ELISA (Human CCL17/TARC DuoSet ELISA, R&D Systems) according to the IFN- duplicate. The lower limits of detection were: IL-5 (0.1 pg/ml), IL-13 (0.4 pg/ml), IL-4 (0.6 pg/ml), in All assays run were (Millipore). immunoassays array multiplex suspension by and supernatants -80°C. at stored and 2 day at collected were Supernatants 18 hours. for ionomycin PMA and γ 3 /150 (0.1pg/mL), GM-CSF (9.5 pg/ml) and eotaxi and pg/ml) (9.5 GM-CSF (0.1pg/mL), Flow-sorted ILC2 cells were cultured in 96-well round bottom plates at a concentration of of plates round bottom a concentration at in96-well ILC2cultured cells were Flow-sorted Geometric means (GM) were used for measurements of central tendency. Group means were meanswere Group tendency. central of measurements used for were (GM) means Geometric Levels IL-4, of IL-5, IL-13,GM-CSF, and eotaxin1/CCL11 were measuredbothinserum

L/well in X-Vivo™ 15 medium (Lonza) supplemented with 1% heat-inactivated human human 1%heat-inactivated with supplemented (Lonza) medium 15 X-Vivo™ in L/well tically significant for all tests. For the microarrayForthe tests. significant forall tically n1/CCL11 (1.2 pg/ml). Serum pg/ml). TARC n1/CCL11 levels were (1.2 α ) levels were quantified in in ) levelsserum were quantified Accepted Article between the two (1/ between thegroups two This by is protectedarticle reserved. Allrights copyright. (1/ affectedfamily members in increased was significantly mRNA expression mean and in5ofGeometric 7healthyin 8of (Figure 9unaffected2A). members family controls of expression constitutive Figure 2, IL5 SPARC MY IL4, FGF1, IL12B, GRIA1, ECSCR,CSF2, genes: thefollowing seen for was family members and unaffected affected between cutoff) 2 of fold-change negative positive or a p=0.05 (after expression thehighest region, differential genes mapped previously inthe 313 setsthe for the totheprobe Restricting analysis 2004). Blood (Klion 5q31-33 chromosome to mapped FEdominant autosomal with family described the previously of 9affected members and 8 unaffected from using RNA PBMC performed analysis was microarray members family affected from PBMC in expression mRNA IL5 Increased Results groups (Supplemental Figure 3). 3). Figure groups (Supplemental of mRNA expression cytokine, cytokine, Th1 the of expression mRNA qRT-PCR. using tested members family unaffected 8 or affected the 8 inany of detectable wasnot the analysis), microarray on members based family affected increased in cytokines, Th2 other two from mRNA contrast, In respectively). p<0.005, and p<0.0001 healthy controls, and in 0.04 in members, 0.03 family unaffected vs. family members mRNA expressionunstimulated in PBMC wasassessed next byqRT-PCR. As canseen be in (Figure 1). (Figure 1). Given the dramatic increase in eosinophilia in FE and the known role of IL-5 in eosinophilia, inFE IL-5 of and therole known ineosinophilia ineosinophilia, Given increase thedramatic FE, in PBMCTo of gene investigate the role driving eosinophilia the expression in

IFN γ , was detectable in 10 affected and 8 unaffected family members, but was comparable comparable was but members, family unaffected 8 and affected 10 in wasdetectable , RAD50 Δ C , , a gene proximity to in close t of 0.05 in affected and unaffected family members) (Figure 2B). family members) affected and unaffected 0.05 in of IL5 mRNA inallwas 10affected detected family members tested, OZ3, MZB1, NRG2, PCDHGC4, PDGFRB, PSD2, PSD2, PDGFRB, PCDHGC4, NRG2, OZ3, MZB1, IL5 , was also comparable thetwo between , comparable was also IL13 Δ and and C t of 0.07 in 0.07 affected in of IL4 IL5 (also , ARHGEF37, ARHGEF37, IL5

Accepted Article This by is protectedarticle reserved. Allrights copyright. sIL5-R and ofIL-5 levels serum Increased tested. Serum levels of TARC/CCL17, a marker of T cell activation, and the Th1 cytokine, IFN- Th1cytokine, and the activation, Tcell of a marker TARC/CCL17, of levels Serum tested. subjects the of any from serum in measurable not were levels IL-4 Serum 3). Figure respectively; andp=0.03, family IU/mL p=0.01 IU/mL members; vs. 19.41 affected 57.23 unaffected in in and of IgE serum pg/mLvs.GM family pg/mL 1.52 members 5.02 level inaffected unaffected in of IL-13 level (GM serum members family affected in decreased IL-13 and of significantly were IgE levels Serum assessed. were levels IgE serum as well as IL-4, and IL-13 cytokine, 2 type the levels of members, p=0.02). family pg/mL unaffected pg/mLvs. 241.2 in (GM 128.6 family members inaffected decreased were eotaxin1/CCL11, also chemokine, the of eosinophil-attracting levels serum Surprisingly, p=0.02). members; family unaffected in pg/mL 1.33 vs. pg/mL 0.37 (GM members family affected 3). between (Figure groups two the were similar to eosinophilia shown serumand has increasebeen ofsIL-5R levels of aplays induction inthe that role cytokine Serum levels GM-CSF, of another members; p<0.01). family unaffected ng/mL in vs.174.9 ng/mL members (GM 450.9 family affected increased in levels of sIL-5R Serum p<0.05). members; family pg/mL 1.60 inunaffected pg/mL vs. family members (GM 4.45 increased inaffected significantly were IL-5 levels meanserum geometric However, exact test). Similar to the mRNAexpression data,serum IL-5 levelswere low, but detectable, Fisher’s (p=NS, members of and in10/14 family the unaffected tested the members affected family inall 13 of To explore whether the increased IL-5 was part of a more generalized Type 2 response, serum serum Type 2response, a of more was part generalized IL-5 increased the To explore whether α , a surrogate marker of cytokine-driven eosinophilia (7), were also significantly were alsosignificantly (7), cytokine-driven eosinophilia of marker , a surrogate α in affected family members family affected in α , was significantly decreased in in decreased significantly was ,

γ , Accepted Article This by is protectedarticle reserved. Allrights copyright. allsubsets). for p<0.05 5B, tested the3 (Figure membersand noneof family members unaffected CD14+ and CD19+ 6affected from all CD8+, atlowlevels, CD4+, flow-sorted family albeit in Furthermore p<0.01). 5A, (Figure members family affected from subsets unaffected family members. and cells and CD3- CD3+ bead-enriched magnetic from prepared cDNAwas Asstep, a next affected and from subsets and CD3-CD19+ CD3-CD14+ CD3+CD8+, CD3+CD4+, flow-sorted from 4). Figure (Supplementary members family unaffected and affected the between detected were differences theobserved increased for mightbe reason the components/subsets PBMCparticular of alterations whether todetermine flow cytometry byassessed blood whole NKT cells were andCD3+CD16+CD56+ NKcells CD3-CD16+CD56+ CD20+, CD3+CD25+, and absolute be IL-5, percentages the of a to source aremajor Since T known CD4+ cells CD3+HLADR+, cells, CD3-CD4- CD8+CD3+, T CD4+CD3+, CD4+, CD8+, numbers CD3+, of restrict not is mRNA IL5 of expression Increased groups (Figure 4). No IL-5, IL-4 or IL-13 was detectable in supernatants from unstimulated PBMC. between two the pg/mL comparable pg/mL13 (GM were 186.5 and302.5 unaffected) in affected in IL-and members) family unaffected pg/mL in vs.191.4 affected in pg/mL 73.46 (GM IL-4 levels of Stimulated PBMC p=0.059). supernatant pg/mL family pg/mL members; vs. 26.49 unaffected in PMA/ionomycin-stimulated be to from higher IL-5 Although the supernatants in tended levels (GM 221.4 significance not reach statistical did difference the affectedPBMC family members, from members Increased release IL-5following PMA/ionomyof IL5 mRNA was significantly upregulated in both CD3+ and CD3- CD3+ both in upregulated mRNA wassignificantly ed CD4+to Tcells affected familymembers in cin stimulation of PBMC from affected family affectedPBMCfamily of from cin stimulation IL5 expression. No significant Nosignificant expression. IL5 mRNA was detectable, mRNAwasdetectable, Accepted Article Although microarray analysis revealed few differences in PBMC gene expression between affected affected PBMCbetween in gene expression differences few revealed analysis microarray Although the indriving eosinophilia. cells study circulating of mononuclear current focused onthe role analyses. additional precluded have constraints logistical and study for available members family affected of number small the identified, been recently has IL-5 of production over asymptomatic and eosinophilia Although a second with family increased 3 generations one largeto kindred. date have to the been restricted the studies isthe fact of study that present The limitation ofmajor eosinophilia. the regulation on 5q31-q33 chromosome genetic in abnormality study a single of to context,effect FE the a In studies. this opportunity provides unique functional of the interpretation complicate a of 2response, Type more generalized and suggestive findings other genetics of the complex Furthermore, associations. these underlying mechanisms specific the about known is little (12), disease cardiovascular of a risk with high adults recently, and, more in (11) schistosomiasis (9,10), children including asthma in settings, other in 5q31-q33 to have chromosome (AEC) counts linked been eosinophil absolute Whereas beenhas thecausativenot identified. mutation (4), mapped 5q31-q33 tochromosome thegene has been Although clinical without consequences. blood theperipheral arein increased Discussion 6B). Figure controls; in healthy 6.194 vs. 185.9 of IL-5 (GM net PMA/ionomycin with stimulation IL-5 following of amounts increased produced members family Fi members; family the affected in 2-10%) (range 4% to compared controls healthy in 2-10%) (range 7% (GM groups thetwo in ILC population total of the proportion a similar for accounted subset ILC2 the Whereas controls (n=6). normal those from and compared to (n=3) family members affected in assessed were ILC subsets CD3- PBMC fraction, This by is protectedarticle reserved. Allrights copyright. Given an thelack theeosinophil to ofevidenceabnormality pointing FE, in lineage in the eosinophils in normal seemingly which (FE) is disorder a eosinophilia genetic Familial rare for increased account the IL-5are could and of Since ILCs source a known gure6A), flow-sorted ILC2s from the affected these conditions IgE and the presence elevated of conditions these

IL5 seen in the the seenin Accepted Article This by is protectedarticle reserved. Allrights copyright. Increased 5). Figure (Supplemental groups two between the similar that expressIL-5R lineages only and theother cells, mast basophils increases in but qRT-PCR.significant note, mild Of in a increase relative members, family and unaffected were significantly decreased in affected family members. inaffected family members. decreased were significantly concomitant increase inPBMC of expression wasno there however, eosinophilia, helminth-driven seen inallergen or responses Th2 generalized serum level andinsupe atthe in significant protein regulators of expression of of expression of regulators selective Nevertheless, (13,14). together expressed are mostoften region chromosome and 5 of some patients with lymphocytic variant HES (20,21). HES (20,21). variant lymphocytic some with patients from IL-2 T cell clonal and in populations with T (19) clones inmurine demonstrated cell stimulated convincingly been only IL-5had of production selective study, present the to prior contrast, In measles with children as in as(16) well individuals cytometry IL-5, 4 and atopic normal in haveflow not by but and IL-13, been demonstrated IL4 region portion (LCR) the 3’ of the gene locus control containing RAD50 (14,22), large geneencoding IL4 elements might lead to isolated expression of of expression might elements isolated to lead upregulation of RAD50 is consistent with the hypothesis that that hypothesis the with consistent is RAD50 of upregulation concomitant of lack the study, present the In (24). mIR-1248 to response in occur to shown hasbeen this dysregulation of expression is lineage-independent. expression islineage-independent. of this dysregulation of levels increased expressed types examined cell all that The fact expression. mechanism for regulatory a the novel of LCR described andsuggests previously and and and and The genes for the three major the three Th2 cytokines, Themajor genes for Whereas coordinated expression of of expression coordinated Whereas IL13 IL13 the to and tothe LCR, separated eachother are from adjacent but . Consequently, it is not difficult to imagine ascenario in which specific regulatory α in humans, were also seen in affected family members. Other lineages were lineages family Other members. inaffected alsoseen inhumans, were RAD50 IL4 . Furthermore, and IL13 have been described (15). Moreover, Th2 cells producing IL- cells Moreover,producing Th2 (15). have beendescribed IL5 IL4 IL5 IL4 transcription occurs in a direction opposite from that of that from opposite a direction in occurs transcription , IL5 (23). In fact, isolated upregulated upregulated isolated (23). Infact, or or rnatants from stimulated PBMCs. Incontrast to the (17) and adult (17) patients with filarial infection (18). and IL13 IL5 IL5 IL13 , mRNA, andIgE andserum IL-13 levels expression was observed and confirmed by confirmed and wasobserved expression IL4 IL5 has been shown to bya regulated be shown to has been and and is not being expressed under control control expressedbeing under is not IL13 IL5 , are clustered in a 160 kb 160 a in clustered are , expression wasalso expression IL5 IL5 IL5

the genes encoding encoding the genes mRNA implies that gene by thesingle IL5 mRNA mRNA transcription Accepted Article 5. Mjösberg JM, Trifari S, Crellin NK, CM, NK, PietB, CP, van Drunen Peters S, Crellin et al. Human IL-25- Mjösberg JM, Trifari 5. Rioux JD, Stone VA, Daly MJ, Cargill M, Green T, etNguyen H, al. Familial eosinophilia 4. ML,W,et al. M, Familial Horne McMaster AD,Law Brown MR, Klion MA, Riemenschneider 3. Naiman JL, Oski FA, Allen FH,LK. Diamond 2. Lin AY, Nutman TB, D, Mulvihill Kaslow JJ, Fontaine L,White BJ, et al. Familial 1. References that made possible. this study care andpatient management in protocol the who have participated Parasitic Diseases of Laboratory of the Section Parasitology the Clinical like of thankto many the The would members authors Acknowledgments disorders. eosinophilic of the treatment for strategies new therapeutic of development the this could lead humans. to Ultimately, in to exploreIL-5 mechanism a of regulation novel opportunity FEa unique provides disorders. in eosinophilic intervention asthma and other therapeutic a target for prime has made it eosinophils andsurvival of activation IL-5 theproduction, in eosinophilia IL-5and increased by peripheral The cells. production mononuclear role blood central of This by is protectedarticle reserved. Allrights copyright. 1998 Oct;63(4):1086–94. Am Hum J Genet. 5q31-q33. onhuman region chromosomal maps cluster tothe cytokine gene 2004 Jun Blood. a 1;103(11):4050–5. eosinophilia: benigndisorder? Jun;16(2):195–203. 1964 Genet. Hum J Am literature. the review of 19;76(3):229–37. clinical Am resultseosinophilia: Med on a Marand laboratory J kindred. U.S. Genet. 1998 and IL-33-responsive type 2 innate lymphoid cells are defined by expression of CRTH2 and of by cells defined expression are lymphoid type2innate and IL-33-responsive by unknown persistent characterized of etiology disorder aIngenetic summary, FE is rare Hereditary eosinophilia: report of report eosinophilia: aHereditaryof family and Accepted Article 11. Rodrigues V, Abel L, Piper K, Dessein AJ. Segregation analysis indicates a major gene in gene athe major in indicates analysis L,Segregation AJ. K,Dessein Piper V,Abel Rodrigues 11. MartinezCJ, Grav S,HolbergFD, Solomon 10. of Linkage WO, et al. Cookson NA,Tay GK, Gibson PJ, LJ,SE, Rye Palmer Daniels 9. Multiple of Rate in False the On theAdaptive Discovery Hochberg Control Y. Y, Benjamini 8. 15. Tanaka S, Motomura Y, Suzuki Y, Yagi R, Inoue H, Miyatake S, et al. The enhancer HS2 Theenhancer al. et Miyatake S, H, Inoue R, Yagi Y, Suzuki Y, Motomura S, Tanaka 15. Flavell Th2cytokine the Regulation RA. TJ, aof by locus Lee Griffin PE, Fields locus GR, 14. in cluster cytokine andIL-5 of the IL-4, regulation IL-13, RM. Locksley Kelly BL, Coordinate 13. Genetic et al. K, P, Gertow H,Almgren E, Björkbacka Valdes-Marquez A, O,Silveira McLeod 12. 7. Wilson TM, Maric I, Shukla J, Brown M, Santos C, Simakova O, et al. IL-5 receptor IL-5 etal. C, receptor Santos M, Brown O, Wilson Simakova J, MaricI, TM, Shukla 7. PepperLE, EK, The Edwards Wilson SD, utilityCJ. Saunders CL, Miller MAS5 of expression 6. This by is protectedarticle reserved. Allrights copyright. Nov;128(5):1086–92.e1. 2011 Immunol. mastocytosis. or marked Clin J Allergy eosinophilia with patients Jan;12(1):77–85. 2011 cells. NatImmunol. inT(H)2 transcription Il4 regulates critically GATA-3-mediated Jul;19(1):145–53. 2003 Immunity. region. control Sep 15;165(6):2982–6. 2000 Immunol. J patterns. expression Th2 allelic by clones revealed May;81:1–9. 2016 Cytokine. disease. cardiovascular with risk a count in for high European population andeosinophil levels interleukin-5 circulating of with loci onchromosome associated are5 Genet.Aug;59(2):453–61. 1996 control of interleukine-5 production in humans infected with Schistosoma mansoni. Am J Hum Dec;158(6):1739–44. Care1998 Crit Med. Am 5q. J Respir onchromosome markers to eosinophils circulating Am Respir Crit J children. Care Dec;158(6):1825–30. Med. 1998 Australian in traits quantitative asthma-associated to 11qgene 5qand chromosome markers 1;25(1):60–83. Jan 2000 Statistics. Educational andBehavioral of Journal Statistics. With Testing Independent 30;8:273. Jul 2007 BMC Bioinformatics. algorithms. call and summary detection 2011 NatImmunol. CD161. Nov;12(11):1055–62. es PE, Baldini M, Erickson RP. Linkage of Linkage of RP. Erickson M, Baldini PE, es α levelsin Accepted Article This by is protectedarticle reserved. Allrights copyright. Panganiban RPL, Pinkerton MH, Maru SY, Jefferson SJ, Roff AN, IshmaelFT. Differential 24. Jan;12(1):16–7. 2011 Immunol. Nat coordination. discordant Explaining M. Bix M, VanStry 23. for LCRLee Lee et Th2 CG, W, KohBH, al. essential Kang JY, SS, M-J, Hwang Kim is 22. interleukin- producing Tcells of clones Abnormal K. Blaser R, Dummer Plötz SG, HU, Simon 21. Th2 K, A, Clonal Kennes B, et al. C, Efira Sibille Willard-Gallo L, F, Roufosse Schandené 20. 4and of5 regulation interleukin interleukin Bohjanen Hodes Okajima Differential PR, M, RJ. 19. Kumaran etal. PP, V, TB, Nutman LE, Hanna PJ, Anuradha George R, Chandrasekaran 18. IL-5, (IL)-4, of interleukin Differential DE. regulation Monze Moss Griffin M, WJ, JJ, Ryon 17. a defines expression IL-5 C. Hierarchical DouekDC, Prussin YinY, Hill BJ, B, Upadhyaya 16. 7;341(15):1112–20. Oct Med. 1999 J N Engl eosinophilia. idiopathic 5 in Jun;109(3):540–8. 2000 Haematol. J Br syndrome. hypereosinophilic idiopathic the with patients in lymphocytes lymphokines. NatlAcad SciU Proc Jul;87(14):5283–7. S A. 1990 exogenous byor antibody byanti-CD3 T-cellgene induction of comparison a expression: gene 2014 Jan 30;8(1):e2658. IL-10 and TGF dependence on differential and their lymphatic filariasis in Th2 human subpopulations IL-5(-) andIL-5(+) of driven expansion Parasite-antigen 1;186(7):879–87. Oct 2002 Dis. Infect J children. Zambian in measles during IL-10 and 20. Sep 15;187(6):3111– 2011 human Immunol. J Th2 cells. highly differentiated of subpopulation Nov15;1(2):154–65. 2012 Immunol. IL-5. of Exp in miR-1248 inasthma JClin of regulation andtherole Am epression microRNA S Jun 8;107(23):10614–9. A. 2010 Acad allergic asthma. of Natl Sci U Proc Th2 of cytokine pathogenesis regulation genes for and β . PLoS Negl Trop Dis. Dis. Trop Negl PLoS . Accepted Article This by is protectedarticle reserved. Allrights copyright. were statistically judged be region the in differences expression the none of Thus, to 0.70. 0.32 from genes ranged these selected Rate valuesThe for False Discovery gene corresponding symbol. are 0.05 labeled p-value their < with and (unadjusted) greater than 2–fold differences expression with sets Probe expression. average geometric in difference for ANOVA test of Results (A) 5q33.1]. - [5q31.1 region chromosomal for analysis expression Gene 1. Figure Figure legends. group. each means for geometric the represent lines The horizontal members. family (UA) and unaffected (A) individual affected for are shown PBMC supernatants and PMA/ionomycin stimulated IL-13 in IL-5, PBMC Levels FE. in IL-4, of mitogen-stimulated from levelsinsupernatants Figure 4. Cytokine **p<0.005 *p<0.05, group. each thegeometric means for lines members.represent The horizontal GM-CSF, TARC/CCL17and IFN- Figure 3. Serum mediator levels in FE. Serum levels ofIL-5, sIL-5R as 0001 family members. toaffected compared and ****p<0. **p<0.005 group. each means for geometric the represent lines Horizontal (HC). andhealthy family members controls Figure 2. Cytokine mRNA expression by PBMC in FE. genes. expressed, differentially most selected, the levels for Individual expression subject (B) applied. wastestingmultiple unstimulated PBMC by unstimulated as qRT-PCR assessed γ are shown for individual affected (A) and unaffected (UA) family (UA) and affected (A) unaffected individual shown for are

is shown as 1/ IL5 , Δ IL4 C

t , for affected (A) and unaffected (UA) (UA) and unaffected affected (A) for significant once this correction for for correction this oncesignificant IL13 α , IL-13, IgE, eotaxin1/CCL11, eotaxin1/CCL11, IgE, IL-13, , and and IFN γ mRNA expression by mRNA expression Accepted Article members. family affected thege lines represent Horizontal subsets. members. B)Net IL-5release from unst in for shown are subsets ILC3 and ILC2 I net and frequency subset ILC 6. Figure toaffected family members. compared li horizontal The family members. (UA) by qRT-PCR asassessed subsets PBMC 5. Figure IL5 mRNA expression byPB expression mRNA M M n n d d L L i i i o i C subsets in FE. FE. in C subsets mulated and PMA/Ionomycin stimulated, flow-sorted flow-sorted stimulated, andPMA/Ionomycin mulated *p< group. each means for the geometric es represent ividual healthycontrols and (HC) affected(A) family s shown as 1/ as shown s -5 production in FE. A) Percentage distribution of IL FE. Percentage distribution in A) -5 production metric means for each group. *p<0.05 as compared t compared as *p<0.05 group. each for means metric Δ C IL5 t for individual affected (A) affected (A) and una individual for mRNA expression by unstimula f f t o 0 C C1, C1, fected ed ed ILC2

.05 as.05

Accepted Article This by is protectedarticle reserved. Allrights copyright.

Accepted Article This by is protectedarticle reserved. Allrights copyright.

Accepted Article This by is protectedarticle reserved. Allrights copyright.