Transcriptional Heterogeneity of Mast Cells and Basophils upon Activation Krishan D. Chhiba, Chia-Lin Hsu, Sergejs Berdnikovs and Paul J. Bryce This information is current as of September 27, 2021. J Immunol published online 5 May 2017 http://www.jimmunol.org/content/early/2017/05/05/jimmun ol.1601825 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2017/05/05/jimmunol.160182 Material 5.DCSupplemental Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 27, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 5, 2017, doi:10.4049/jimmunol.1601825 The Journal of Immunology Transcriptional Heterogeneity of Mast Cells and Basophils upon Activation Krishan D. Chhiba, Chia-Lin Hsu, Sergejs Berdnikovs, and Paul J. Bryce Mast cells and basophils are developmentally related cells whose activation is a hallmark of allergy. Functionally, mast cells and basophils overlap in their ability to produce several mediators, including histamine and granule proteases, but studies have increas- ingly demonstrated nonredundant roles. To characterize the transcriptional heterogeneity of mast cells and basophils upon their activation, we performed large-scale comparative microarrays of murine bone marrow–derived mast cells and bone marrow– derived basophils (BMBs) at rest, upon an adaptive-type activation (IgE cross-linking), or upon an innate-type activation (IL-33 stimulation). Hierarchical clustering demonstrated that bone marrow–derived mast cells and BMBs shared specific activation- associated transcriptional signatures but differed in other signatures both between cell type and between activation mode. In bone marrow–derived mast cells, IgE cross-linking upregulated 785 genes, including Egr2, Ccl1, and Fxyd6, whereas IL-33 Downloaded from stimulation induced 823 genes, including Ccl1, Egr2, and Il1b. Focused bioinformatics pathway analysis demonstrated that IgE activation aligned with processes such as oxidative phosphorylation, angiogenesis, and the p53 pathway. The IL-33–activated transcriptome was enriched in genes commonly altered by NF-kB in response to TNF, by IL-6 via STAT3, and in response to IFN-g. Furthermore, BMBs activated via IgE cross-linking selectively induced immune response genes Ccl1, Il3, and Il2 compared with IL-33–stimulated BMBs. Principal-component analysis revealed key cell- and activation-specific clustering. Overall, our data demonstrate that mast cells and basophils have cell- and activation-specific transcriptional responses and suggest that context- http://www.jimmunol.org/ specific gene networks and pathways may shape how the immune system responds to allergens and innate cytokines. The Journal of Immunology, 2017, 198: 000–000. ast cells and basophils are critical effector cells in type to months. Conversely, mature basophils circulate in the blood be- I immediate hypersensitivity reactions and associate fore being recruited to tissues and have a shorter lifespan (60–70 h). M with allergic disease pathology. The developmental As a result of their developmental similarity, circulating basophils pathways of mast cells and basophils are closely related but are also are often used as surrogates to study tissue-resident mast cell re- the subject of continuing controversy (1). In the bone marrow, mast activity [e.g., the basophil activation test for diagnosing food al- cells and basophils are commonly considered to begin as common lergy–associated reactivity (7)], but the similarity or heterogeneity by guest on September 27, 2021 myeloid progenitor cells, progress to granulocyte-monocyte pro- between the two cell types is relatively unknown. genitors, and become granulocyte progenitors. Functionally, gran- Generally, mast cells and basophils respond to many of the same ulocyte progenitors have been shown to differentiate into basophils stimuli, and their functional roles appear to overlap. The best- or mast cell progenitors (MCPs) (2, 3). However, this model has studied shared mechanism of activation between the cell types is been challenged by studies at the single-cell level that define MCP cross-linking of the FcεRI receptor, which promotes the release of potential within a common myeloid population and not in the histamine, as well as other granule mediators, eicosanoids, and granulocyte/macrophage progenitor population (4). Common pro- inflammatory cytokines. In addition to activation through FcεRI, genitors are also found in the spleen and can generate basophils and mast cells and basophils express ST2, the receptor for IL-33. MCPs (5, 6). MCPs are released into the circulation and mature IL-33 is an “epithelial-derived” cytokine that promotes type once they are in tissues; mature mast cells have a lifespan of weeks 2–associated immune responses and has been strongly linked to allergy (8). Upon IL-33–mediated activation, mast cells have been shown to exhibit enhanced adhesion, survival, maturation, and Division of Allergy and Immunology, Department of Medicine, Northwestern Uni- production of several proinflammatory cytokines (9, 10). In ba- versity Feinberg School of Medicine, Chicago, IL 60611 sophils, IL-33–mediated activation has been described as pro- ORCID: 0000-0002-6997-7667 (K.D.C.). moting migration toward eotaxin and enhancing degranulation Received for publication October 25, 2016. Accepted for publication April 10, 2017. upon concurrent IgE-mediated activation (11). For cytokine pro- This work was supported by National Institutes of Health Grant R01AI105839 (to P.J.B.). duction, basophils were also shown to respond poorly to IL-33 The microarray data presented in this article have been submitted to the National compared with IgE-mediated activation, but a relatively small Center for Biotechnology Information’s Gene Expression Omnibus (https://www. number of mediators were studied (12, 13). Therefore, defining ncbi.nlm.nih.gov/geo/) under accession number GSE96696. the transcriptional activation signatures of mast cells and baso- Address correspondence and reprint requests to Dr. Paul J. Bryce, Division of phils using a systems biology–based approach is likely to help Allergy and Immunology, Feinberg School of Medicine, Northwestern University, 240 E. Huron Street, Chicago, IL 60611. E-mail address: [email protected] understand the possible contributions of each cell type and the The online version of this article contains supplemental material. mode of activation to the progression of allergic diseases. Abbreviations used in this article: AREG, amphiregulin; BMB, bone marrow–derived Recent evidence has shown that resting mast cells and basophils basophil; BMMC, bone marrow–derived mast cell; GSEA, Gene Set Enrichment actually have relatively low transcriptional homology in mice and Analysis; log2FC, log2 fold change; m, mouse; MCP, mast cell progenitor; PCA, humans (14, 15), implying that they might be much more distinct principal component analysis; ST2KO, ST2-knockout; WT, wild-type. than once thought. However, the similarities and/or differences in Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 their transcriptional signatures upon activation are unknown. It is www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601825 2 MAST CELL AND BASOPHIL EXPRESSION SIGNATURES well appreciated that mast cells have functions in health and using an Agilent 2100 Bioanalyzer. cDNA was synthesized with qScript disease. In line with their role as sentinel cells, mast cells encode cDNA Supermix (Quanta Biosciences). Gene expression was verified for pathways for the synthesis of a diverse array of mediators. They select genes by real-time PCR using an ABI 7500 instrument and TaqMan probes (both from Applied Biosystems). are critical players in the symptoms of anaphylaxis, mediate re- sistance to infection, and promote tumor rejection. Relatively less Microarray experiments and statistical analyses is known about basophil functions. Basophils rapidly secrete IL-4 RNA was hybridized to Illumina MouseWG-6 v2.0 arrays (catalog number and IL-13 upon IgE-mediated activation, play a role in the host BD-201-0202). Preprocessing steps, including quality control, background defense against parasites, and promote Th2 responses through adjustment, and quantile normalization, were performed using the array altering Ag presentation (13, 16). Although mast cells and baso- analysis tools available at http://www.arrayanalysis.org using the lumi phils are believed to have nonredundant roles in immune regula- package and bgAdjust function (18). Variance stabilization was managed with a log2 transformation. Normalized data were filtered to remove unex- tion, the evidence supporting this idea has been scarce until now, pressed probes and beads with a detection p value , 0.01. Groups