Human Eosinophils Express a Distinct Gene Expression Program In

Human Eosinophils Express a Distinct Gene Expression Program in Response to IL-3 Compared with Common β-Chain Cytokines IL-5 and GM-CSF This information is current as of October 4, 2021. Ryan K. Nelson, Howard Brickner, Bharat Panwar, Ciro Ramírez-Suástegui, Sara Herrera-de la Mata, Neiman Liu, Damaris Diaz, Laura E. Crotty Alexander, Ferhat Ay, Pandurangan Vijayanand, Grégory Seumois and Praveen Akuthota Downloaded from J Immunol 2019; 203:329-337; Prepublished online 7 June 2019; doi: 10.4049/jimmunol.1801668 http://www.jimmunol.org/content/203/2/329 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Human Eosinophils Express a Distinct Gene Expression Program in Response to IL-3 Compared with Common b-Chain Cytokines IL-5 and GM-CSF

Ryan K. Nelson,*,† Howard Brickner,*,† Bharat Panwar,† Ciro Ramı´rez-Sua´stegui,† Sara Herrera-de la Mata,† Neiman Liu,*,† Damaris Diaz,*,† Laura E. Crotty Alexander,*,‡ Ferhat Ay,†,x Pandurangan Vijayanand,† Gre´gory Seumois,†,1 and Praveen Akuthota*,†,1

Despite recent advances in asthma management with anti–IL-5 therapies, many patients have eosinophilic asthma that remains poorly controlled. IL-3 shares a common b subunit receptor with both IL-5 and GM-CSF but, through a-subunit–specific properties, uniquely influences eosinophil biology and may serve as a potential therapeutic target. We aimed to globally characterize the transcriptomic profiles of GM-CSF, IL-3, and IL-5 stimulation on human circulating eosinophils and identify Downloaded from differences in gene expression using advanced statistical modeling. Human eosinophils were isolated from the peripheral blood of healthy volunteers and stimulated with either GM-CSF, IL-3, or IL-5 for 48 h. RNA was then extracted and bulk sequencing performed. DESeq analysis identified differentially expressed genes and weighted gene coexpression network analysis indepen- dently defined modules of genes that are highly coexpressed. GM-CSF, IL-3, and IL-5 commonly upregulated 252 genes and downregulated 553 genes, producing a proinflammatory and survival phenotype that was predominantly mediated through TWEAK signaling. IL-3 stimulation yielded the most numbers of differentially expressed genes that were also highly coexpressed http://www.jimmunol.org/ (n = 119). These genes were enriched in pathways involving JAK/STAT signaling. GM-CSF and IL-5 stimulation demonstrated redundancy in eosinophil gene expression. In conclusion, IL-3 produces a distinct eosinophil gene expression program among the b-chain receptor cytokines. IL-3–upregulated genes may provide a foundation for research into therapeutics for patients with eosinophilic asthma who do not respond to anti–IL-5 therapies. The Journal of Immunology, 2019, 203: 329–337.

sthma is a chronic disease of the airway associated with exacerbation rates, reduced oral glucocorticoid use, improved lung significant morbidity. In the United States alone, costs function, and improved asthma control scores in randomized control related to asthma have amounted to more than 80 billion trials (2, 3). Nevertheless, not all patients with eosinophilic asthma A by guest on October 4, 2021 dollars annually (1). Although a significant proportion of asth- respond to anti–IL-5 therapies clinically, and populations of func- matics maintain control via inhaled corticosteroid therapy, disease tional eosinophils have been shown to remain in the airway despite heterogeneity has caused many individuals to remain poorly con- anti–IL-5 treatment (4, 5). It has, therefore, been suggested that trolled with either high symptom scores and/or frequent exacerba- alternative mechanisms remain relevant to the pathogenesis of eo- tions. The recent approval of mAbs against IL-5 has improved sinophilic asthma in certain individuals and that further investiga- control of severe asthmatics with eosinophilia, demonstrating lower tion is necessary to optimize precision treatment strategies. One area of investigative focus has involved the cytokines IL-3 and GM-CSF. Similar to IL-5, these two cytokines are produced as *Division of Pulmonary, Critical Care, and Sleep Medicine, University of California part of the Th2 inflammatory response and are crucial to eosino- † San Diego, La Jolla, CA 92037; La Jolla Institute for Immunology, La Jolla, CA phil development and function (6, 7). Despite all three cytokines 92037; ‡Veterans Affairs San Diego Healthcare System, La Jolla, CA 92161; and x b University of California San Diego School of Medicine, La Jolla, CA 92093 sharing a common -chain receptor subunit, each differentially 1G.S. and P.A. are joint senior authors. affects eosinophil biology as the result of divergent downstream intracellular signaling events through a-chain subunit-specific ORCIDs: 0000-0003-0075-7399 (H.B.); 0000-0001-8126-710X (C.R.-S.); 0000- 0002-9519-9520 (S.H.-d.l.M.); 0000-0002-7266-2404 (N.L.); 0000-0002-3347- properties (6, 8). Of these cytokines, IL-3 has been shown to 9578 (D.D.); 0000-0002-5091-2660 (L.E.C.A.); 0000-0002-0708-6914 (F.A.); most strongly and differentially affect eosinophil function, espe- 0000-0002-8164-6852 (G.S.). cially over prolonged stimulation periods (6). This, together with Received for publication December 26, 2018. Accepted for publication May 15, the observation that eosinophils recruited to the airway following 2019. allergen challenge have increased surface levels of the a-chain This work was supported by National Institutes of Health Grants NIH K08HL116429 a (to P.A.) and NIH S10OD016262 (to G.S.). subunit specific to IL-3, but reduced levels of the -chain subunit The sequences presented in this article have been submitted to the Gene Expression specific to IL-5, has made IL-3 an attractive potential target in Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE128027. asthma therapeutics (5, 9). Although knowledge of eosinophil Address correspondence and reprint requests to Dr. Praveen Akuthota, University of biology as it relates to IL-3 stimulation has been rapidly growing, California San Diego, 9500 Gilman Drive, MC 7381, La Jolla, CA 92037. E-mail no study to date has used large-scale gene expression profiling to address: [email protected] define the IL-3 signature in eosinophils. In the present work, we The online version of this article contains supplemental material. use whole-transcriptome bulk RNA sequencing methods and ad- Abbreviations used in this article: TPM, transcripts per million; WGCNA, weighted vanced statistical modeling to demonstrate a unique transcrip- gene coexpression network analysis. tional profile of human blood eosinophils stimulated by IL-3 ex Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 vivo after being harvested from normal subjects. Because IL-3 has www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801668 330 EOSINOPHIL b-CHAIN CYTOKINE DIFFERENTIAL GENE EXPRESSION been previously described to prolong intracellular signaling in removal of such duplicates can worsen the power and false discovery eosinophils (6), we specifically report results following cytokine rate for differential gene expression (15, 16). The sequences presented stimulation for 48 h. in this article have been submitted to Gene Expression Omnibus (http:// www.ncbi.nlm.nih.gov/geo/) under accession number GSE128027. Materials and Methods RNA sequencing analysis Patient characteristics RNA sequencing data were mapped against the hg38 reference using Venous blood specimens were obtained after informed consent under a TopHat (17) (v1.4.1.,–library-type fr-secondstrand -C) and the RefSeq protocol approved by the Institutional Review Board at the University of gene annotation downloaded from the University of California, Santa Cruz California San Diego. Whole blood (160 ml) was drawn from 13 healthy Genome Browser site. The read coverage per gene was computed using volunteers without known allergic disease (seven for gene expression HTSeq-count (-m union -s yes -texon -i gene_id,http://www-huber.embl. studies and six for flow cytometry experiments). de/users/anders/HTSeq). To identify genes differentially expressed between GM-CSF, IL-3, and IL-5–stimulated, as well as unstimulated eo- Eosinophil isolation and stimulation sinophils, negative binomial tests for pairwise comparisons employing the Bioconductor package DESeq2 (v1.16.1) were performed (18). Genes Eosinophils were purified from the peripheral blood of normal donors by were considered differentially expressed between any pairwise comparison negative selection (StemCell Technologies, Vancouver, Canada) as previ- when DESeq2 analysis resulted in a Benjamini–Hochberg-adjusted p value ously described (10). Briefly, RBCs were depleted by hetastarch incubation , 0.01 (1% false discovery rate) and Log2 (fold change) $ 1. Moreover, followed by gravity separation. Granulocytes were isolated by centrifu- any gene identified as commonly upregulated or commonly downregulated gation of RBC-depleted blood over a Ficoll gradient. Eosinophils were passed this threshold in all individual pairwise comparisons. Pathway then isolated from the granulocyte fraction by incubation with a mixture of analysis was performed using Ingenuity Pathway Analysis (Qiagen) (19). negative selection Abs followed by passage over a magnetized column. Eosinophil purity was routinely .99% by Hema 3 staining (Thermo Fisher Weighted gene coexpression network analysis Scientific, Medford, MA), and eosinophil viability was routinely .99% Downloaded from by trypan blue exclusion after cell isolation. Purified eosinophils from Weighted correlation network analysis using the R package weighted gene each donor were split equally into unstimulated, GM-CSF–stimulated, coexpression network analysis (WGCNA; version 1.61) was performed on , IL-3–stimulated, and IL-5–stimulated groups (1–2 3 106 eosinophils per the transcripts per million data (TPM) matrix (20). Values 10 TPM in group). Human rIL-3, rIL-5 (R&D Systems, Minneapolis, MN), and all samples were removed, as established in the third phase of the GM-CSF (BioLegend, San Diego, CA) were used for stimulation, all for MicroArray Quality Control project, also called Sequencing Quality 48 h at a concentration of 10 ng/ml in RPMI 1640 (supplemented with 10% Control (21). A total of 11,889 well-expressed genes were used for generating the coexpression network. A b = 10 was selected following the FBS and 1% penicillin/streptomycin) at 37˚C. Cell viability remained .90% http://www.jimmunol.org/ for all conditions after 48 h of stimulation, as assessed by propidium iodide scale-free topology criterion (22). Gene modules were generated using staining. For gene expression experiments, eosinophils were resuspended in a blockwiseModules function (parameters: checkMissingData = TRUE, phenol/guanidine-based lysis reagent (QIAzol; Qiagen), either immediately power = 10, TOMType = unsigned, minModuleSize = 30, maxBlockSize = after purification for unstimulated cells or after 48 h of stimulation, and 11,889, and mergeCutHeight = 0.25). A total of 24 different modules were stored at 280˚C prior to RNA extraction. For flow cytometry experiments, generated, including a gray module for non-coexpressed genes, which was unstimulated eosinophils were stored at 4˚C prior to staining, and stimulated excluded from further analysis. Because each module, by definition, is cells were stained after 48 h. Unstimulated eosinophils remained .98% comprised of highly correlated genes, their combined expression may be viable after storage at 4˚C for 48 h. usefully summarized by eigengene profiles, effectively the first principal component of a given module. A small number of eigengene profiles RNA extraction may, therefore, summarize the principle patterns within the cellular tran-

scriptome with minimal loss of information. This dimensionality-reduction by guest on October 4, 2021 Isolated and stimulated eosinophils from seven donors were stored in approach facilitated calculation of a Spearman correlation for modules and QIAzol Lysis Reagent as previously described. Total RNA was isolated using each clinical trait (unstimulated, stimulated, IL-3, IL-5, and GM-CSF) miRNeasy Micro Kit (Qiagen) loaded on an automated platform (QIAcube; used in the analysis. Qiagen). Samples were quantified as described previously (11, 12), and To visualize coexpression networks, the function exportNetworkToCytoscape quality of RNA assessed by Fragment Analyzer (Advance Analytical). All at weighted = true and threshold = 0.05 was used. A soft thresholding power . samples had an RNA integrity number 8.0 and passed our quality and was chosen based on the criterion of approximate scale-free topology. quantity control steps as described previously (12, 13). Networks were generated in Gephi (version 0.9.2) using the Fruchterman mRNA sequencing library preparation Reingold layout algorithm, followed by Noverlap to eliminate individual node overlap (23). The size of each gene node was scaled according to the Purified total RNA (∼5 ng) was amplified following the Smart-seq2 pro- average degree as calculated in Gephi. tocol (13, 14). Briefly, mRNA was captured using poly-dT oligos and di- rectly reverse transcribed into full-length cDNA using the described Flow cytometry template-switching oligo (13, 14). cDNA was amplified by PCR for CD69 and CD131 (CSF2RB) were selected as proteins of interest based 14–15 cycles and purified using AMPure XP magnetic bead (0.9:1 [vol:vol] on the RNA sequencing analysis presented in this manuscript and their ratio; Beckman Coulter). From this step, for each sample, 1 ng of cDNA was expression was measured by flow cytometry. Peripheral eosinophils from used to prepare a standard NextEra XT sequencing library (NextEra XT six donors were isolated as described above and stained using specific DNA Library Prep Kit and Index Kits; Illumina). Barcoded Illumina se- fluorescently conjugated Abs: mouse anti-human CD69 coupled to APC/Cy7 quencing libraries (Nextera; Illumina) were generated using an automated P (clone FN50; BioLegend) and mouse anti-human CD131 coupled with PE platform (Biomek FX ; Beckman Coulter). Both whole-transcriptome am- (clone 1C1; BioLegend). Providing three biologic replicates, stimulated plification and sequencing library preparations were performed in a 96-well eosinophils from three donors were surface stained with anti-CD69. Similarly, format to reduce assay-to-assay variability. Quality control steps were stimulated eosinophils from three separate donors were permeabilized using included to determine total RNA quality and quantity, the optimal number 100% methanol and stained with anti-CD131 (CSF2RB). Flow cytometry of PCR preamplification cycles, and fragment library size (13). Samples data were obtained with a BD Biosciences LSR II, gated to eosinophils that failed quality controls were eliminated from downstream steps. Li- using forward and side scatter characteristics in this already-highly purified braries that passed strict quality controls were pooled at equimolar con- population, and analyzed with FlowJo 10.5.0 (Tree Star, Ashland, OR). centration, loaded, and sequenced on the Illumina Sequencing platform, HiSeq2500 (Illumina). Libraries were sequenced to obtain more than 8 million 50-bp, single-end reads (HiSeq Rapid Run Cluster and Sequencing Results by Synthesis Kit V2; Illumina) mapping uniquely to mRNA reference, b-Chain receptor cytokines share common eosinophil generating a total of ∼252 million mapped reads (median of ∼8million filtered mapped reads per sample). Approximately 40% of the total reads activation signals were duplicates. Given the inability to separate artifactual read duplicates We first aimed to categorize the transcriptomic profiles distinguishing from true biological duplicates in RNA sequencing (unlike in DNA se- b quencing, in which the initial number of copies is known), we did not filter eosinophils stimulated with -chain receptor cytokines from those out duplicate reads during analysis. This approach is consistent with prior left unstimulated ex vivo. Principle component analysis of the studies that show many duplicate reads reflect biological reality and that 2000 most variable genes showed clear separation of unstimulated The Journal of Immunology 331 eosinophils from the GM-CSF–, IL-3–, and IL-5–stimulated identified by WGCNA, five modules were highly correlated with groups (Fig. 1A). DESeq2 analysis, which assesses for differences b-chain receptor cytokine stimulus groups (Fig. 2B; r . 0.5; p , 0.01). in average gene expression across groups, identified differentially The two strongest correlations occurred in relation to IL-3 and are expressed genes based on pairwise comparisons among stimulated represented by the pink and yellow modules (Fig. 2B). These two and unstimulated conditions (Benjamini–Hochberg-adjusted p value modules contained 82% of the IL-3–upregulated genes identified , 0.01). To identify a gene expression profile specific to the by DESeq2 analysis, with most of such genes belonging to the stimulated condition (b-chain receptor cytokine-specific genes), we yellow module (Fig. 2C). Interestingly, the hub genes of the yel- identified genes for which all pairwise comparisons between the low module were predominantly genes deemed IL-3 specific by stimulated and unstimulated groups met statistical significance with DESeq2 analysis (Fig. 3, Supplemental Table III, blue nodes/font). a Benjamini–Hochberg-adjusted p value , 0.01 and Log2 (fold Hub genes are the genes that are most tightly coexpressed with change) $ 1. Because DESeq2 does not account for absolute other genes within a given module and, therefore, are thought to transcript levels and because genes exhibiting low expression under be key regulators of their corresponding module’s biology. The stimulated conditions are less likely to have a meaningful biologic green-yellow and red modules (correlating most strongly with effect, we eliminated any gene with ,10 TPM in a stimulated IL-5) and the magenta module (correlating strongly with both group. This analysis revealed 252 genes commonly upregulated and GM-CSF and IL-5) also revealed module-specific hub genes, but 553 genes commonly downregulated by b-chain receptor cytokines without an overlapping, group-specific signal per DESeq2 analysis (Fig. 1B, Supplemental Table I). (Fig. 3). Pathway analysis of the commonly upregulated genes revealed To define the biology of IL-3 stimulation on eosinophils, we multiple pathways using NF-kB signaling, notable for promot- focused on the 119 genes that are both differentially upregulated ing cell survival and proliferation as well as immune mediated (DESeq2) and highly coexpressed (yellow module) (Figs. 2C, 4A). Downloaded from inflammation (24). Of these, the TWEAK (TNFSF12) signal- The top hub genes in the yellow module, defined by a module ing pathway was most significant (Benjamini–Hochberg-adjusted membership r $ 0.95, included 17 genes that were also IL-3 p value = 5.49 3 1027) (Fig. 1C). TWEAK is a cytokine belonging to specific by differential expression (Fig. 4A, Table I). Included in the TNF superfamily and can either be weakly proapoptotic by sig- this cohort were transcripts encoding the common b-chain re- naling through Death Receptor 3 (DR3/TNFRSF25) or prosurvival ceptor subunit for GM-CSF, IL-3, and IL-5 (CSF2RB); surface and inflammatory by signaling through Fibroblast Growth Factor- activation markers CD69 and CD180; two transcriptional regula- http://www.jimmunol.org/ Inducible 14 (Fn14/TNFRSF12A) (25). We discovered upregula- tors extensively linked to the biology of regulatory T cells (IKZF4 tion of TNFRSF12A transcripts as well as its downstream signaling and BACH2); a sulfhydryl oxidase responsible for posttransla- mediators (TRAF3) and effectors (NFKB1/NFKB2) (26). BIRC3, tional disulfide bond formation (QSOX1); a regulator of lysosomal encoding an inhibitor of apoptosis along the TWEAK pathway, dynamics (TBC1D15); and a transcription factor required for early was additionally upregulated. Finally, genes commonly upregu- eosinophil differentiation (XBP1) among other protein-coding lated in response to cytokine stimuli were overrepresented in genes with either known or undefined functions (27–33). FACS several pathways independent of NF-kB signaling, with the top analysis corroborated our sequencing data at the protein level five pathways including the unfolded protein response (CD82, for two of these genes: CD69 and CD131 (CSF2RB), and mean by guest on October 4, 2021 CEBPG, INSIG1, NFE2L2, and SREBF2), phagosome maturation fluorescence intensity for both markers met statistical significance (CTSC, CTSD, CTSL, RAB5A, TUBA1B, TUBA1C, and TUBB4B), by one-way ANOVA (p , 0.05) (Fig. 5). Given that CD69 is a tyrosine degradation (FAH and HPD), granulocyte adhesion and surface marker of leukocyte activation, FACS was performed for diapedesis (CCL1, CCL22, CCL24, CXCL1, ICAM1, IL1A, and surface expression of this protein, whereas whole-cell protein IL1B), and 14-3-3–mediated signaling (ELK1, RRAS, TUBA1B, expression was interrogated for CD131 to ensure that any intra- TUBA1C, TUBB4B, and YWHAG) (Supplemental Table II). cellular stores were assayed. Pathway analysis of the commonly downregulated genes showed an Subsequent pathway analysis of all 119 genes connected to IL-3 overrepresentation of genes involved in IFN signaling (Fig. 1D). We by both DESeq2 and WGCNA revealed an overrepresentation of further discovered downregulation of several proapoptotic genes, genes involved in JAK/STAT signaling as well as of genes linked to including those encoding for the TNF family ligands TNFSF10 and apoptosis mechanisms (Fig. 4B, Supplemental Table IV). The FAS, CASP3 and the PARP family cleavage products, and the latter finding contrasts with the collective prosurvival signal we transcription factor FOXO3 (Supplemental Table II). Collectively, observed in cytokine-stimulated eosinophils as a whole. Three b-chain receptor cytokines stimulate eosinophils to promote a noncoding RNAs (AC090152.1, AC078846.1, and LINC01943) proinflammatory and prosurvival phenotype. were part of this 119-gene group and were, therefore, not recognized by pathway analysis. IL-3 stimulation yields a unique eosinophil gene expression signature Discussion Similarly focusing on genes that maintained significance in dif- This study is the first, to our knowledge, to use two advanced, ferential expression across all pairwise comparisons, we next aimed independent statistical models to analyze whole-transcriptome to categorize cytokine-specific signals. Supported by IL-3 sepa- RNA sequencing and categorize the downstream effects of ration in the principle component analysis (Fig. 1A), IL-3 stim- b-chain receptor cytokine signaling. Using an unbiased ulation resulted in the most unique gene expression profile with pathway analysis approach, we discovered an overrepresen- 158 upregulated genes and 36 downregulated genes relative to all tation of upregulated prosurvival genes common to all three other conditions including unstimulated (Fig. 2A, Supplemental b-chain receptor cytokines. Genes involved in TWEAK sig- Table I). IL-5 analysis revealed one unique downregulated gene naling pathway were most enriched. Although TWEAK/Fn14 (PMAIP1 ), but no upregulated genes. GM-CSF did not produce interactions have been connected to several inflammatory dis- any unique differentially expressed genes in either direction. eases, including asthma; direct evidence supporting its role in WGCNA, which assigns genes into modules based on similar eosinophils is lacking (34, 35). A recent study, however, linked patterns of change in expression across samples, yielded further single-nucleotide polymorphisms in BIRC3, the inhibitor of support for an IL-3–specific gene expression signal. Of the 24 modules apoptosis associated with the TWEAK pathway, with reduced 332 EOSINOPHIL b-CHAIN CYTOKINE DIFFERENTIAL GENE EXPRESSION Downloaded from http://www.jimmunol.org/ by guest on October 4, 2021

FIGURE 1. b-Chain receptor cytokines share common activation signals on eosinophils. (A) t-Distributed stochastic neighbor embedding (tSNE) plot of the top 2000 differentially expressed genes (five principle components; nine perplexity) (B). Heatmap depicting the 252 commonly upregulated and 553 commonly downregulated genes in response to b-chain receptor cytokine stimulation. (C and D) Top 10 pathways represented by the commonly upregulated and downregulated genes. The number of genes identified by differential expression analysis in relation to the total number of genes known for any given pathway are represented by percentages. asthma susceptibility and reduced loads of circulating eosinophils which b-chain receptor cytokines promote eosinophil’s role in (36). Furthermore, BIRC3 has been identified as a potential patho- asthma pathogenesis. genic gene in childhood asthma based on a molecular interaction Most striking in our analysis was the gene expression profile network study (37). produced by IL-3. Consistent with prior work, we found that IL-3 Downregulated genes common to all three b-chain receptor stimulation most uniquely influences eosinophil gene expres- cytokines were similarly enriched in several pathways, with the sion relative to the other b-chain receptor cytokines following most significant pathway related to IFN signaling. INFa has been prolonged stimulation. Using two independent techniques, we demonstrated to inhibit airway eosinophilia and hyperresponsiveness identified a cohort of 119 genes separating IL-3 based both on as well as inhibit eosinophil mediator release (38, 39). This finding, transcript expression levels and coexpression networks. The top together with our observed downregulation of several proapoptotic genes in this cohort included a range of genes with previously genes, supports a role for inhibition of apoptosis as one method by described roles in eosinophils, with defined roles in other cell The Journal of Immunology 333 Downloaded from http://www.jimmunol.org/ by guest on October 4, 2021

FIGURE 2. IL-3 stimulation yields a unique eosinophil gene expression signature. (A) Heatmap depicting the 158 upregulated and 36 downregulated genes in response to IL-3 stimulation. (B) Select WGCNA modules reaching significance (p , 0.01). Numerical cells indicate the Spearman correlation coefficient of the stimulus group to the module. (C) Genes similarly identified by DESeq2 and WGCNA analysis.

lines, but with undescribed function in eosinophils, or with subjects may not reflect the true biology of airway eosinophils undescribed functions altogether. For instance, XBP1 has been in asthma patients. In different tissue compartments, eosino- demonstrated as essential for early eosinophil development, but phils exist in subsets, each displaying distinct phenotypes and less is known about its role in eosinophils that have fully ma- gene expression patterns (46). Although the role of IL-3 tured in the blood or airway (33). QSOX1 has gained interest in stimulation of eosinophils in the airway remains an area of cancer research as a disulfide bond catalyst with an atypical active investigation, there is reason to believe its role is im- localization to the Golgi apparatus and extracellular space (30, portant, particularly in patients who do not respond to anti– 31). The importance of such an enzyme in eosinophils may IL-5 therapy. Not only does IL-3 increase in the airway of relate to eosinophil disulfide bond–containing secretory proteins asthmatic subjects following allergen challenge, eosinophils (e.g., eosinophil peroxidase and major basic protein) with cy- also display increased levels of surface IL-3R following totoxic and immunomodulatory roles in asthma (40–42). Two mepolizumab therapy (5, 47). Esnault et al. (48) previously transcription factors (IKZF4 and BACH2) have been connected published results of microarray analyses on human BAL to regulatory T cell stability, with the latter only indirectly and sputum cells following allergen challenge to the lung. linked to eosinophils in the current literature (27–29, 43). The Additionally, they assessed for gene downregulation follow- interaction between CD69 and its ligands (myosin light chains ing mepolizumab therapy. Among others, they discovered 9 and 12; the former is also a member of our IL-3–defining gene upregulation in genes that are also members of our IL-3– cohort) has been shown to be crucial in allergic airway in- defining cohort. CD69, RHOH,andST6GAL1 were upregu- flammation and the recruitment of activated T cells to sites of latedinbothBALandsputum eosinophils subjected to inflammation (44). Finally, upregulation of the common b-chain allergen challenge; IL2RA, NDFIP2, PIM2, SLC2A1, SOCS1, receptor subunit (CSF2RB) may provide a contributing mech- and SVIP were upregulated in the BAL samples; and CYTIP, anism by which IL-3 uniquely prolongs intracellular signaling IL1RAP, MAST4, OSBPL3, OSM,andSOCS2 were upregu- in eosinophils (6, 45). lated in the sputum samples. Of these genes, CD69, NDFIP2, We acknowledge that these 119 genes identified by cyto- and SOCS1 were the only to decrease following mepolizumab kine stimulation of peripheral blood eosinophils in nonasthmatic treatment. 334 EOSINOPHIL b-CHAIN CYTOKINE DIFFERENTIAL GENE EXPRESSION Downloaded from

FIGURE 3. Hub genes of the yellow WGCNA module include a large number of differentially expressed, IL-3–speciﬁc genes.

Gene connectivity plots of the five significant http://www.jimmunol.org/ WGCNA modules along with the mean gene expression for the most representative (hub) gene of each module. Top 10% of genes from each module are shown. Large nodes indicate hub genes of increased connectivity. Blue nodes represent genes that were group-specific based on DESeq2 analysis. For example, in the yellow module nodes that are depicted in blue rather than yellow represent IL-3–specific genes based on DESeq2 analysis. In this particular module, by guest on October 4, 2021 there were a large number of IL-3–specific, differentially expressed genes among the WGCNA hub genes. Error bars indicate SEM.

We believe the value of our study lies in its genome-wide and make novel connections with either IL-3 or the eosinophil cell and unbiased approach. Such methodology allowed us to iden- type. Furthermore, we linked the expression of a few noncoding tify protein-coding genes, some previously associated with asthma, RNAs to b-chain receptor cytokine stimuli and foresee potential The Journal of Immunology 335 Downloaded from http://www.jimmunol.org/ by guest on October 4, 2021

FIGURE 4. IL-3 stimulation yields 119 differentially expressed and highly coexpressed genes. (A) Genes both differentially upregulated (DESeq2 analysis) and highly coexpressed (yellow module) following IL-3 stimulation (n = 119). (B) Pathway enrichment analysis of these 119 genes. implications as their gene regulatory and epigenetic roles become cytokines simultaneously, at varying concentrations over time. further deﬁned. These coding and noncoding genes may provide Our study of peripheral blood eosinophils from healthy subjects the foundation for future investigation on therapeutic targets in may additionally not account for the effects of the local tissue eosinophilic asthma, and with the recognized increase in IL-3Ra environment or of the host. Finally, we are limited by the lack of expression in airway eosinophils, may address the current short- posttranscript analysis, as prior work has described IL-3–dependent comings of anti–IL-5 therapy. Limitations of our study include the translational modiﬁcations that would be missed in the present snapshot picture that does not account for the presence of multiple work (49). 336 EOSINOPHIL b-CHAIN CYTOKINE DIFFERENTIAL GENE EXPRESSION

Table I. IL-3–speciﬁc hub genes

Gene Signiﬁcance (IL-3) Module Membership Normalized Mean Counts

Gene Symbol GS (r) p Value MM (r) p Value GM-CSF IL-3 IL-5 UNSTIM TBC1D15 0.82 1.06 3 1027 0.98 5.06 3 10219 33 93 22 13 CD180 0.81 1.65 3 1027 0.97 4.33 3 10218 48 287 6 2 CD69 0.82 8.02 3 1028 0.97 5.24 3 10218 580 2445 187 48 IL1RAP 0.77 1.75 3 1026 0.97 1.23 3 10217 54 169 35 26 IKZF4 0.85 1.30 3 1028 0.97 8.24 3 10217 17 69 7 1 QSOX1 0.90 9.91 3 10211 0.96 2.89 3 10216 55 137 42 14 ARRDC4 0.82 1.09 3 1027 0.96 3.87 3 10216 144 546 51 55 PHTF2 0.91 3.48 3 10211 0.96 6.65 3 10216 70 159 56 17 SOS1 0.84 2.10 3 1028 0.96 8.23 3 10216 30 89 25 10 KIAA0040 0.79 7.47 3 1027 0.95 3.74 3 10215 34 120 23 12 BACH2 0.79 6.24 3 1027 0.95 3.79 3 10215 2142 1 FRMD4B 0.73 9.13 3 1026 0.95 8.55 3 10215 15 69 11 3 CSF2RB 0.86 5.37 3 1029 0.95 1.90 3 10214 2332 5110 1804 513 PRNP 0.86 6.73 3 1029 0.95 2.19 3 10214 135 598 104 38 XBP1 0.86 3.20 3 1029 0.95 2.90 3 10214 241 572 151 96 ZNF589 0.79 5.07 3 1027 0.95 3.52 3 10214 9336 6 CYTIP 0.87 1.25 3 1029 0.95 3.64 3 10214 756 1554 567 248 Downloaded from Top hub genes of the yellow module as defined by a correlation coefficient $ 0.95. All 17 genes were also IL-3 specific based on DESeq2 analysis. GS, gene significance; MM, module membership; r, correlation coefficient; UNSTIM, unstimulated.

In summary, these results highlight the redundant cytokine several genes strongly correlated with IL-3 may provide the signaling mechanisms involved in eosinophil development and foundation for future therapeutic advancements in eosinophilic function but support a unique role for IL-3. The identiﬁcation of asthma. http://www.jimmunol.org/ by guest on October 4, 2021

FIGURE 5. Protein levels of CD69 and CD131 in human eosinophils. FACS of surface CD69 and total cell CD131 (CSF2RB) with mean ﬂuorescence intensity (MFI) for each cytokine-stimulated condition. Both met statistical signiﬁcance by one-way ANOVA (p , 0.05). Corresponding sequencing data for each gene are included in TPM. The Journal of Immunology 337

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