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Transcriptional Heterogeneity of Mast Cells and 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

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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 © 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 . Functionally, mast cells and basophils overlap in their ability to produce several mediators, including and , 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 –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, , 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 responds to and innate . 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 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 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 - 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, , and granulocyte/ 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” 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 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 , 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 were and the distinct contributions of each cell type at rest and upon compared using the limma adapted t test. Differentially expressed genes activation are beginning to be appreciated. were identified using the Benjamini–Hochberg false discovery rate correc- To better understand the functional differences and similarities tion (p value # 0.05). Heat maps were generated using GENE-E software (Broad Institute; http://broadinstitute.org/cancer/software/GENE-E). between mast cells and basophils, we performed a large-scale comparison of the transcriptomes of murine mast cells and ba- Cell-specific signature validation sophils at rest and upon activation by an adaptive-type (IgE cross- For each sample, the purity of each BMMC culture was determined to be linking) or innate-type (IL-33) stimulation. Using homogeneous .98% FcεRI+/CD117+, and each BMB culture was .98% FcεRI+/ 2 populations of mature mast cells and basophils derived from bone CD117 /CD49b+. To assess the specificity of the cellular transcriptome Downloaded from marrow, we also asked whether IgE- and IL-33–mediated activa- between our BMMC and BMB populations, we initially examined key tion induced similar or distinct gene signatures in both cell types. genes that were described as reflecting a mast cell–specific signature in a recent study from the Immunological Genome Project Consortium (15), as Through this nonbiased, large-scale approach, we provide evi- well as genes for two basophil proteases that are not expressed in mast dence for heterogeneity between mast cell and cells: Mcpt8 (Mcpt8) and Prss34 (Mcpt11) (19). As shown in Fig. 1D, all responses, as well as in the activation of each individual cell type three mast cell cultures clustered together, whereas the basophil cultures also showed distinct clustering. Furthermore, the mast cell signature was by an innate- or adaptive-type stimuli. The cell- and activation- http://www.jimmunol.org/ specific signatures identified in this study define novel gene net- highly reflective of the mast cell cultures, whereas basophils possessed a unique signature driven by Mcpt8 and Prss34. Additionally, there was no works and pathways that may contribute to understanding how the significant expression of Mitf, a transcription factor described as important immune system responds to allergens and innate cytokines. for early commitment of basophil and mast cell lineages (20).

Materials and Methods Principal component analysis and network diagrams Mice Functional gene set enrichment analysis was used to generate functional gene networks and identify hub genes by meta-grouping of individual gene term sets C57BL/6J mice (4–6 wk old) were obtained from the Jackson Labora- (referencing Gene Ontology Biological Process and Kyoto Encyclopedia of tory. ST2-knockout (ST2KO) mice were previously obtained from Genes and Genomes Pathways) based on function similarity. The GeneTerm by guest on September 27, 2021 Dr. A. McKenzie and backcrossed to C57BL/6J mice for eight generations. Linker algorithm, implemented in the FGNet R package, was used for this All animal studies were performed under Institutional Animal Care and analysis (parameters set for analysis: adjusted p value , 0.05; minimum Use Committee guidelines and under protocols that have been approved by support of three). Networks generated within this analysis were exported in the Northwestern University Animal Care and Use Committee. The bone graph modeling language format for further analysis and visualization using marrow from each individual mouse was used to generate independent the iGraph R package. Cytoscape 3.2.1. was used to visualize individual cultures of mast cells and basophils, as outlined below. metagroups and stimulus-specific versus shared network components. Prin- cipal component analysis (PCA) was performed using the Population PCA Bone marrow–derived mast cells program (Scott Davis, Harvard Medical School) on the top 15% variable $ Bone marrow–derived mast cells (BMMCs) were obtained by flushing bone transcripts with a relative expression 120. Gene Set Enrichment Analysis marrow from femurs and tibias of mice. Cells were cultured in BMMC media (GSEA; Broad Institute) was used to determine enriched hallmark gene sets in activated mast cells and basophils (21). Circos visualization and enrichment (RPMI 1640 media containing 2 mM L-glutamine, 10% FBS [Atlanta Bio- heat maps were generated using http://metascape.org (22). logicals], 25 mM HEPES [Sigma], 1 mM sodium pyruvate [Sigma], 0.1 mM nonessential amino acids [Sigma], 100 U/ml penicillin, 100 mg/ml strepto- mycin [Corning], 0.05 mM 2-ME [Sigma]) and 30 ng/ml recombinant mouse Results (m)IL-3 (Miltenyi Biotec) for 4–6 wk. Purity was assessed by flow cytometry using PE–anti-mFcεRI (MAR-1; eBioscience) and allophycocyanin–anti- IgE- and IL-33–activated mast cells are transcriptionally mCD117 (2B8; BD Biosciences) (Fig. 1A). For IgE/Ag activation, BMMCs distinct were primed with 1 mg/ml anti-OVA–IgE overnight and activated with 0.5 mg/ml OVA (Sigma) for 4 h, as previously described (17). For IL-33 Mast cells and basophils are developmentally related, but they activation, BMMCs were activated with 10 ng/ml IL-33 for 4 h (Fig. 1B). perform similar, as well as nonredundant, roles in allergic disease. One way to determine the degree of differences in their output Bone marrow–derived basophils responses to adaptive (IgE cross-linking) and innate (IL-33) stimuli Bone marrow–derived basophils (BMBs) were generated by flushing bone is to use a nonbiased bioinformatics approach to interrogate the marrow from femurs and tibias of mice. Cells were cultured in BMMC media genetic changes that occur upon activation. Before performing a plus 30 ng/ml recombinant mIL-3 (Miltenyi Biotec) for 9 d. On day 9, sus- cell-specific comparison between mast cells and basophils, we pended cells were sorted using autoMACS (Miltenyi Biotec), first with a negative depletion using mCD117 MicroBeads and then by positive selection asked whether the activation-specific transcriptome was distinct with mCD49b (DX-5) MicroBeads (both from Miltenyi Biotec). Purity of the between IgE and IL-33 activation in each cell type. For mast cell sorted cells was confirmed using flow cytometry for allophycocyanin– activation, we set up an in vitro model using BMMCs, a well- anti-mCD49b (HMa2; BD Biosciences), FITC–anti-mFcεRI (MAR-1; eBio- validated technique for generating a homogeneous mast cell science), and PE–anti-mCD117 (2B8; BioLegend) (Fig. 1A). IgE/Ag and IL-33 . activation of BMBs was performed as described above for BMMCs (Fig. 1C). population, with an average purity 98% (Fig. 1A) (23). BMMCs have properties of serosal- and mucosal-type mast cells and are RNA isolation and real-time PCR extensively used to study FcεRI signaling (24). Using array-based RNA was isolated from unstimulated and stimulated cells with an RNeasy technology, we analyzed the expression of .45,000 transcripts in RNA Isolation Kit (QIAGEN). RNA quality assessment was performed unstimulated and IgE/Ag- or IL-33–stimulated BMMCs in tripli- The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 1. Generation of murine mast cells and basophils. (A) Bone marrow cells were cultured in rIL-3 (30 ng/ml) for 4 wk to generate mast cells (BMMCs). Basophils (BMBs) were purified from the bone marrow culture after 10 d with rIL-3 by an initial negative depletion for mCD117, followed by positive selection with mCD49b magnetic beads. Cell surface FcεRI, CD117, or CD49b expression was used to validate purity. BMMCs (B) and BMBs (C) were left unstimulated or were activated with IgE cross-linking or IL-33 stimulation in triplicate. (D) RNA from these samples was sent for microarray analysis, and expression of key mast cell and basophil genes in the WT unstimulated samples was used to initially verify that cell-specific signatures were evident. cate, with each culture generated from individual mice. The For IgE activation, cross-linking of anti-OVA–IgE with OVA for microarray data presented in this article have been submitted to 4 h resulted in 785 differentially expressed genes (∼4.0%) com- the National Center for Biotechnology Information’s Gene Ex- pared with unstimulated mast cells (adjusted p value # 0.05). A pression Omnibus under accession number GSE96696 (https:// heat map of upregulated and downregulated genes was generated www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE96696). by filtering (log2 fold change [log2FC] . 2, average expression 4 MAST CELL AND BASOPHIL EXPRESSION SIGNATURES

$ 5, and p # 0.05) and then ranking the top 50 transcripts transcriptome. In fact, of the ∼45,000 analyzed transcripts, pair- according to their adjusted p value (Fig. 2A). From these top 50 wise comparison between the groups found that only Rpe was genes, we observed three major patterns of gene expression: genes significantly upregulated (4.9-fold) in WT BMMCs. downregulated by IgE activation, genes upregulated by IgE acti- We next performed a pairwise comparison of IL-33–stimulated vation, and genes upregulated by IgE and IL-33 activation. Of the versus –unstimulated WT and ST2KO mast cells and identified differentially expressed genes, 309 genes were upregulated $2- 823 differentially expressed genes (∼4.2%) that were statistically fold, including Il33, as our laboratory had described previously significant after 4 h. A heat map of the top 50 upregulated or (17). In contrast, 234 genes were downregulated $2-fold, illus- downregulated genes illustrates two main subsets of transcripts: trated by the balanced appearance of significant data points on the those induced by IL-33 but not IgE and those induced by both volcano plot (Fig. 2B). Among the genes with the highest fold stimuli (Fig. 3A). Notably, the IL-33–induced genes remained change were Egr2 (72.26-fold), Ccl1 (67.24-fold), and Fxyd6 unchanged in ST2KO BMMCs upon IL-33 stimulation. Pairwise (62.30-fold) (Fig. 2C, 2D). Wild-type (WT) and ST2KO BMMCs comparison of unstimulated WT BMMCs and IL-33–stimulated that underwent IgE-mediated activation had virtually the same ST2KO BMMCs again identified only Rpe as having a statistically Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 2. Transcriptional signature of IgE-activated mast cells. (A) Heat map of the top 50 significantly changed genes in BMMCs after IgE-mediated activation. (B) Volcano plot showing log10(p value) versus log2FC. Colored points represent p , 0.05 (red), log2FC . 2.5 (orange), and p , 0.05 and log2FC . 2.5 (green). Top 10 upregulated (C) and downregulated (D) genes after IgE-mediated activation, ranked by fold change. The Journal of Immunology 5 significant change (upregulated by 6.4-fold). A volcano plot of the in response to TNF, upregulated by IL-6 via STAT3, and upreg- IL-33–induced transcriptome displays a rightward shift, with ulated in response to IFN-g (Supplemental Fig. 1B). A network many more upregulated genes (log2FC .2.5) than downregulated diagram constructed from significantly upregulated genes revealed genes (Fig. 3B). IL-33 stimulation resulted in 299 upregulated and gene signatures that were unique and common to these two stimuli 248 downregulated genes (by $2-fold); the genes with the greatest (Fig. 4). As we have reported previously and now confirm with fold change compared with unstimulated mast cells were Ccl1 this microarray approach, the induction of Il33 expression by mast (138.59-fold), Egr2 (104.90-fold), and Il1b (62.56-fold) (Fig. 3C, 3D). cells is a signature unique to IgE-mediated activation (Fig. 4) (17). Using GSEA, we analyzed the top differentially expressed hallmark gene sets enriched in activated mast cells. IgE–cross- Basophils are strongly activated by IgE-mediated, but not linked mast cells induced genes that encode for that IL-33–mediated, stimulation participate in oxidative phosphorylation, angiogenesis, and p53 To determine the innate-type and adaptive-type activation tran- pathways (Supplemental Fig. 1A). In contrast, IL-33–stimulated scriptome of basophils, we derived the transcriptional signature of mast cells induced genes that are commonly regulated by NF-kB activated BMBs (average purity . 98%) in response to IgE cross- Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 3. Mast cell transcriptional signature after IL-33 activation. (A) Heat map of the top 50 significantly changed genes in BMMCs after IL-33 stimulation. (B) Volcano plot showing log10(p value) versus log2FC. Colored points represent p , 0.05 (red), log2FC . 2.5 (orange), and p , 0.05 and log2FC . 2.5 (green). Top 10 upregulated (C) and downregulated (D) genes after IL-33 stimulation, ranked by fold change. 6 MAST CELL AND BASOPHIL EXPRESSION SIGNATURES Downloaded from http://www.jimmunol.org/

FIGURE 4. Network diagram of upregulated genes in IgE-activated or IL-33–stimulated mast cells.

linking and IL-33. IgE-mediated activation resulted in 3986 dif- IgE-activated basophils: MYC-regulated genes (subgroup v1), genes by guest on September 27, 2021 ferentially expressed genes (20.5%) that achieved statistical upregulated during the unfolded response, and genes up- significance with an adjusted p value # 0.05. The heat map of the regulated by STAT5 in response to IL-2 stimulation (Supplemental top 50 upregulated and downregulated genes included many Fig. 1E). In contrast, IL-33–stimulated transcripts aligned with the transcripts encoding secreted cytokines and (Fig. 5A). following hallmark genes sets: genes upregulated by KRAS activa- Like in BMMCs, we observed Il33 induction upon IgE cross- tion, genes defining the inflammatory response, and genes regulated linking but not after IL-33 stimulation. A volcano plot of the by NF-kB in response to TNF (Supplemental Fig. 1F). data from IgE stimulation demonstrates a wide spread of data points away from the origin that results from many transcripts Mast cells and basophils are transcriptionally distinct from each other at rest and after stimulation having a large log2FC and a low p value (Fig. 5B). Of these displayed transcripts, 875 distinct genes increased by $2-fold, and As a final step in our analyses, we quantified the differences between 730 genes decreased by $2-fold. The genes with the highest fold mast cells and basophils at rest and after activation by comparing the change were upregulated Ccl1 (369.39-fold), Il3 (222.99-fold), lists of differentially expressed transcripts in all four groups from the and Il2 (107.41-fold) (Fig. 5C, 5D). pairwise comparisons illustrated above. Using a Circos plot, we il- In response to IL-33 stimulation, WT, but not ST2KO, basophils lustrate matching genes among treatments and cell types by drawing displayed altered expression of 238 genes (1.2%) with statistical purple lines to link identical genes in two given groups (Fig. 8A). The significance (142 were upregulated and 5 were downregulated $2- dark orange areas on the inner arc represent the subset of genes fold). The heat map displays the top 39 genes expressed by IL-33– found in at least one other group, whereas the light orange areas stimulated basophils (Fig. 6A). Unlike the previous groups, the signify the proportion of genes that are unique to the given group. A pairwise comparison of IL-33–stimulated basophils and unstimu- Venn diagram illustrating the specific numbers of overlapping versus lated basophils resulted in ,50 transcripts that met the cut-offs: unique genes between each group is shown in Fig. 8B, and a list of log2FC . 2, average expression $ 5, and p # 0.05. The rightward these genes is provided in Supplemental Table I. Surprisingly, only bias of the volcano plot illustrates that IL-33 increases the ex- two genes (Tnf and Traf1) were common to both cell types and both pression of most genes and decreases relatively few (Fig. 6B). The stimuli, and the numbers of unique genes within each specific cell- top three differentially regulated genes in IL-33–stimulated ba- stimulus category dominated over those that were shared across sophils were Plat (73.18-fold), Cxcl2 (22.24-fold), and Tpbg categories. PCA using the top 15% of transcripts with the most (19.54-fold) (Fig. 6C, 6D). variability (6473 probes) illustrates cell type–attributable variation A network diagram constructed from the upregulated transcripts captured by PC1 and PC2, as well as stimulation-specific variation illustrates the groups of genes and pathways that appear to be IgE along PC3 (Fig. 8C). A subset of genes representing the cell and specific, IL-33 specific, and common responses to stimuli (Fig. 7). By stimulus-specific signatures was validated in independent cultures GSEA, we identified the three most enriched hallmark gene sets in using real-time RT-PCR methods (Supplemental Fig. 2). The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 5. Basophil transcriptional signature after IgE activation. (A) Heat map of the top 50 significantly changed genes in BMBs after IgE-mediated activation. (B) Volcano plot showing log10(p value) versus log2FC. Colored points represent p , 0.05 (red), log2FC . 2.5 (orange), and p , 0.05 and log2FC . 2.5 (green). Top 10 upregulated (C) and downregulated (D) genes after IgE-mediated activation, ranked by fold change.

Taken together, our data suggest that the core transcriptional scriptome of mast cells and basophils at rest and upon activation with response to innate- and adaptive-type stimuli can be defined. We an innate type (IL-33) or adaptive type (IgE cross-linking) of stimuli. analyzed transcriptional responses in mast cells and basophils and We use IL-33 as the innate stimuli, because it is well known to activate define the cell-specific signature for these two stimuli. From the cell- mast cells through ST2, it promotes type 2 immune responses (9, 10), and activation-specific profiles, we derive novel gene networks and and basophils also express ST2 and have been shown to respond to pathways that may participate in how the immune system responds IL-33 (13, 25). To address the question above, we used a nonbiased, to allergens and innate cytokines. bioinformatics approach that provided a global analysis of the tran- scriptional changes that occurredinmastcellsandbasophilsupon Discussion activation. Through PCA, we confirmed that mast cells and basophils Mast cells and basophils are developmentally related cells that play have distinct transcriptional programs at rest and further defined that key effector roles in allergic and nonallergic diseases, and the extent of they also have distinct transcriptional programs upon activation with their functional similarities and differences in response is not yet fully an adaptive- or innate-type stimulation (Fig. 8C). Also, through an- understood. In this article, we broaden our understanding of these two alyzing IgE–cross-linked and IL-33–stimulated mast cells and baso- cell types by asking whether heterogeneity exists between the tran- phils independently, we surprisingly observed that transcriptional 8 MAST CELL AND BASOPHIL EXPRESSION SIGNATURES Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 6. Basophil transcriptional signature after IL-33 activation. (A) Heat map of the top 50 significantly changed genes in BMBs after IL-33 stimulation. (B) Volcano plot showing log10(p value) versus log2FC. Colored points represent p , 0.05 (red), log2FC . 2.5 (orange), and p , 0.05 and log2FC . 2.5 (green). Top 10 upregulated (C) and downregulated (D) genes after IL-33 stimulation, ranked by fold change. differences were enhanced after activation (even with the same IgE-activated mast cells in our arrays; this finding may suggest a stimuli), suggesting even further functional differences. role for basophil-derived chemokines in CD4+ regulation. Focusing on mast cells, IgE-activated mast cells shared a small A surprising aspect of our findings is that basophils respond transcriptional signature (327 genes) with IL-33–stimulated mast strongly to IgE-mediated activation and less potently to IL-33, unlike cells. Using this shared signature, we identified the hallmark gene the balanced activation signatures seen with mast cells. Only 20 sets enriched after both modes of activation: processes associated differentially expressed genes were shared between the two activation with allograft rejection, apoptosis, and cholesterol homeostasis groups in basophils. Basophils activated via IgE cross-linking (Supplemental Fig. 1D). Interestingly, activation of mast cells exhibited the most robust response among all of the tested groups, through FcεRI or ST2 dramatically increased expression of Ccl1 with 20.5% of the transcriptome altered upon activation, including and Egr2. CCL1 is a that is important for regulating genes regulated by MYC (subgroup v1 and v2), as well as genes immune cell migration, specifically CD4+ T cell trafficking (26, participating in oxidative phosphorylation and the unfolded protein 27). Other groups have reported the Egr2-dependent induction of response. In contrast, the IL-33–altered genes in basophils reflected CCL1 in mast cells after IgE-mediated activation (26–28), con- quite unexpected pathways, including heme metabolism, mitotic necting these molecules to a shared regulatory pathway; however, spindle assembly, and adipogenesis. The PCA plots further highlight production of CCL1 in mast cells after IL-33 stimulation has not the divergence of response phenotype in basophils, with IgE- been described previously. Furthermore, based on fold-change activated basophils of both genotypes (WT and ST2KO) clustering values, IgE–cross-linked basophils expressed more Ccl1 than did tightly with one another but distantly from all other groups, including The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/

FIGURE 7. Network diagram of upregulated genes in IgE-activated or IL-33–stimulated basophils. by guest on September 27, 2021

IL-33–activated basophils. Interestingly, we do show that amphir- formed IL-33, the potential remains for a later influence of in- egulin (AREG), a growth factor in the epidermal growth factor duced IL-33 being secreted and functioning in an autocrine fashion. family that is known to be involved in allergic responses, is induced Although a recent study by Dwyer et al. (15) derives mast in basophils by both IgE cross-linking and IL-33 (Fig. 5A). Previ- cell– and basophil-specific transcriptional signatures at rest, our ously, it was reported that human basophils produced AREG in re- work extends these findings by characterizing the responses of sponse to IL-3, and the investigators suggested that basophil-derived these cell types after activation by innate- and adaptive-type AREG contributed to tissue remodeling and repair (29, 30). Recently, stimuli. Indeed, this comparative analysis of mast cells and ba- AREG derived from type 2 innate lymphoid cells was shown to sophils after activation provides additional evidence that these mediate their tissue-protective function in intestinal tissue (31). cells may independently regulate immune responses during active In this article, we provide the first evidence, to our knowledge, to disease. This finding challenges the general assumption that cir- suggest that activated basophils could participate in similar tissue- culating basophils can be used as functional surrogates for tissue- remodeling processes during type 2 immune responses in allergic resident mast cells and, at the very least, highlights the need for diseases through their expression of AREG. caution in doing so in research or clinical practice. In this study, we include ST2-deficient cells and observe virtually A potential caveat in our study is that the use of bone marrow– no significant transcriptional differences compared with WT cells at derived primary cells might not fully extrapolate in phenotype to rest and upon IgE-mediated activation. The lack of a response in tissue-derived cells, as was the approach used by Dwyer et al. (15) for ST2KO mast cells and basophils to IL-33 illustrates the necessity of the Immunological Genome Project Consortium. However, two key the ST2 receptor and the specificity of the IL-33–activation signa- points can be raised when considering our findings within the context ture. Because we previously demonstrated that IgE cross-linking of that previous study. First, as we show in Fig. 1, the mast cells promoted IL-33 expression in mast cells (17, 32) (and now also generated for our study demonstrate a clear preservation of their in basophils, as shown above), we postulated that preformed or reported “mast cell signature,” indicating that BMMCs reflect a core induced IL-33 could feed back through the ST2 receptor to po- mast cell transcriptomic profile and not that of a precursor or other tentiate cellular activation. Because the IgE-activated transcriptome lineage cell. Second, using bone marrow–derived primary cells ac- was identical in WT and ST2KO BMMCs and BMBs, this type of tually provides us with the distinct advantage of profiling transcrip- autocrine regulation of IL-33/ST2 signaling, as has been proposed tional responses in highly homogeneous populations of mast cells and to occur in dendritic cells (33), seems unlikely. However, a caveat to basophils, because they have been generated under similar culture and this concept is that we investigated only one time point (4 h); thus, activation conditions; moreover, these cells were generated without although our findings seem to rule out any contribution of pre- using techniques that could unintentionally activate the cells, such as 10 MAST CELL AND BASOPHIL EXPRESSION SIGNATURES Downloaded from

FIGURE 8. Comparison of the transcriptional signature of mast cells and basophils after activation. (A) Circos plot showing overlap among filtered genes for http://www.jimmunol.org/ each group (log2FC . 2, average expression $ 5, and p # 0.05). Purple curves link identical genes. Light orange areas within the inner arc indicate unique genes. Dark orange areas within the inner arc represent genes that are differentially expressed in another group. (B) Venn diagram illustrating the frequency of overlapping and unique genes among groups. (C) PCA of the population using the top 15% probes with the most variable expression. Ba, basophil; MC, mast cell. enzymatic disruption or mechanical separation. Indeed, Dwyer et al. 4. Franco, C. B., C. C. Chen, M. Drukker, I. L. Weissman, and S. J. Galli. 2010. (15) actually reported upregulation of several key genes upon diges- Distinguishing mast cell and granulocyte differentiation at the single-cell level. Cell Stem Cell 6: 361–368. tion enzyme exposure that we demonstrate in this article as reflecting 5. Arinobu, Y., H. Iwasaki, M. F. Gurish, S. Mizuno, H. Shigematsu, H. Ozawa, activation signatures, including Egr2 and Ccl1, implicating basal D. G. Tenen, K. F. Austen, and K. Akashi. 2005. Developmental checkpoints of activation of endogenous mast cells during the methods currently used the basophil/mast cell lineages in adult murine hematopoiesis. Proc. Natl. Acad. by guest on September 27, 2021 Sci. USA 102: 18105–18110. to extract them from tissues. For this reason, it is likely impossible to 6. Chen, C. C., M. A. Grimbaldeston, M. Tsai, I. L. Weissman, and S. J. Galli. discriminate stimulus-specific signatures using tissue-isolated mast 2005. Identification of mast cell progenitors in adult mice. Proc. Natl. Acad. Sci. cells with the clarity that we have been able to achieve in this study USA 102: 11408–11413. 7. Santos, A. F., and G. Lack. 2016. Basophil activation test: food challenge in a using a homogeneous bone marrow–derived cell population. test tube or specialist research tool? Clin. Transl. Allergy 6: 10. In conclusion, we demonstrate that mast cells and basophils have 8. Grotenboer, N. S., M. E. Ketelaar, G. H. Koppelman, and M. C. Nawijn. 2013. cell- and activation-specific transcriptional signatures. The limited Decoding : translating genetic variation in IL33 and IL1RL1 into disease . J. Allergy Clin. Immunol. 131: 856–865. homology that we observe between these cell types after activation 9. Iikura, M., H. Suto, N. Kajiwara, K. Oboki, T. Ohno, Y. Okayama, H. Saito, with the same stimuli suggests that these cells play independent S. J. Galli, and S. Nakae. 2007. IL-33 can promote survival, adhesion and cy- roles in the immune response. These findings provide a firm tokine production in human mast cells. Lab. Invest. 87: 971–978. 10. Saluja, R., M. Khan, M. K. Church, and M. Maurer. 2015. The role of IL-33 and foundation upon which future investigations can build on our mast cells in allergy and inflammation. Clin. Transl. Allergy 5: 33. collective understanding of functional differences between these 11. Suzukawa, M., M. Iikura, R. Koketsu, H. Nagase, C. Tamura, A. Komiya, developmentally related cell types. S. Nakae, K. Matsushima, K. Ohta, K. Yamamoto, and M. Yamaguchi. 2008. An IL-1 cytokine member, IL-33, induces human basophil activation via its ST2 receptor. J. Immunol. 181: 5981–5989. Acknowledgments 12. Silver, M. R., A. Margulis, N. Wood, S. J. Goldman, M. Kasaian, and D. Chaudhary. 2010. IL-33 synergizes with IgE-dependent and IgE-independent We thank Dr. Andrew McKenzie for kindly providing the ST2KO mice, the agents to promote mast cell and basophil activation. Inflamm. Res. 59: 207–218. Northwestern University Feinberg School of Medicine NUSeq core facility for 13. Yoshimoto, T., K. Yasuda, H. Tanaka, M. Nakahira, Y. Imai, Y. Fujimori, and running the microarrays, Dr. Matthew Schipma for bioinformatics and pathway K. Nakanishi. 2009. Basophils contribute to T(H)2-IgE responses in vivo via analysis guidance, and Dr. Mendy Miller for writing and editing assistance. IL-4 production and presentation of peptide-MHC class II complexes to CD4+ T cells. Nat. Immunol. 10: 706–712. 14. Motakis, E., S. Guhl, Y. Ishizu, M. Itoh, H. Kawaji, M. de Hoon, T. Lassmann, Disclosures P. Carninci, Y. Hayashizaki, T. Zuberbier, et al. 2014. Redefinition of the human The authors have no financial conflicts of interest. mast cell transcriptome by deep-CAGE sequencing. Blood 123: e58–e67. 15. Dwyer, D. F., N. A. Barrett, and K. F. Austen, Immunological Genome Project Consortium. 2016. Expression profiling of constitutive mast cells reveals a unique identity within the immune system. Nat. Immunol. 17: 878–887. References 16. Stone, K. D., C. Prussin, and D. D. Metcalfe. 2010. IgE, mast cells, basophils, 1. Sasaki, H., D. Kurotaki, and T. Tamura. 2016. Regulation of basophil and mast and . J. Allergy Clin. Immunol. 125(2, Suppl. 2): S73–S80. cell development by transcription factors. Allergol. Int. 65: 127–134. 17. Hsu, C. L., C. V. Neilsen, and P. J. Bryce. 2010. IL-33 is produced by mast cells 2. Iwasaki, H., S. Mizuno, Y. Arinobu, H. Ozawa, Y. Mori, H. Shigematsu, and regulates IgE-dependent inflammation. PLoS One 5: e11944. K. Takatsu, D. G. Tenen, and K. Akashi. 2006. The order of expression of 18. Eijssen, L. M., M. Jaillard, M. E. Adriaens, S. Gaj, P. J. de Groot, M. Muller,€ and transcription factors directs hierarchical specification of hematopoietic lineages. C. T. Evelo. 2013. User-friendly solutions for microarray quality control and pre- Genes Dev. 20: 3010–3021. processing on ArrayAnalysis.org. Nucleic Acids Res. 41: W71–W76. 3. Iwasaki, H., and K. Akashi. 2007. Myeloid lineage commitment from the he- 19. Ugajin, T., T. Kojima, K. Mukai, K. Obata, Y. Kawano, Y. Minegishi, Y. Eishi, matopoietic stem cell. Immunity 26: 726–740. H. Yokozeki, and H. Karasuyama. 2009. Basophils preferentially express mouse The Journal of Immunology 11

mast cell 11 among the mast cell family in contrast to mast volvement of mast cells and T cells in allergic mucosal inflammation: critical cells. J. Leukoc. Biol. 86: 1417–1425. role of the CC chemokine ligand 1:CCR8 axis. J. Immunol. 179: 1740–1750. 20. Qi, X., J. Hong, L. Chaves, Y. Zhuang, Y. Chen, D. Wang, J. Chabon, B. Graham, 27. Wu, Z., A. J. Macneil, R. Junkins, B. Li, J. N. Berman, and T. J. Lin. 2013. Mast K. Ohmori, Y. Li, and H. Huang. 2013. Antagonistic regulation by the tran- cell FcεRI-induced early growth response 2 regulates CC chemokine ligand scription factors C/EBPa and MITF specifies basophil and mast cell fates. Im- 1-dependent CD4+ T cell migration. J. Immunol. 190: 4500–4507. munity 39: 97–110. 28. Vaali, K., J. Lappalainen, A. H. Lin, M. I. Ma¨yra¨npa¨a¨, P. T. Kovanen, A. Berstad, 21. Subramanian, A., P. Tamayo, V. K. Mootha, S. Mukherjee, B. L. Ebert, and K. K. Eklund. 2012. mesylate alleviates diarrhea in a mouse model M. A. Gillette, A. Paulovich, S. L. Pomeroy, T. R. Golub, E. S. Lander, and J. P. Mesirov. 2005. Gene set enrichment analysis: a knowledge-based approach of intestinal allergy. Neurogastroenterol. Motil. 24: e325–e335. for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. USA 29. Qi, Y., D. J. Operario, C. M. Oberholzer, J. J. Kobie, R. J. Looney, S. N. Georas, 102: 15545–15550. and T. R. Mosmann. 2010. Human basophils express amphiregulin in response to 22. Tripathi, S., M. O. Pohl, Y. Zhou, A. Rodriguez-Frandsen, G. Wang, D. A. Stein, T cell-derived IL-3. J. Allergy Clin. Immunol. 126: 1260–1266.e1264. H. M. Moulton, P. DeJesus, J. Che, L. C. Mulder, et al. 2015. Meta- and or- 30. Meulenbroeks, C., H. van Weelden, C. Schwartz, D. Voehringer, F. A. Redegeld, thogonal integration of influenza “OMICs” data defines a role for UBR4 in virus V. P. Rutten, T. Willemse, A. J. Sijts, and D. M. Zaiss. 2015. Basophil-derived budding. Cell Host Microbe 18: 723–735. amphiregulin is essential for UVB irradiation-induced immune suppression. 23. Tertian, G., Y. P. Yung, D. Guy-Grand, and M. A. Moore. 1981. Long-term J. Invest. Dermatol. 135: 222–228. in vitro culture of murine mast cells. I. Description of a growth factor- 31. Monticelli, L. A., L. C. Osborne, M. Noti, S. V. Tran, D. M. Zaiss, and D. Artis. dependent culture technique. J. Immunol. 127: 788–794. 2015. IL-33 promotes an innate immune pathway of intestinal tissue protection 24. Wershil, B. K., and S. J. Galli. 1994. The analysis of mast cell function in vivo dependent on amphiregulin-EGFR interactions. Proc. Natl. Acad. Sci. USA 112: using mast cell-deficient mice. Adv. Exp. Med. Biol. 347: 39–54. 10762–10767. 25. Schneider, E., A. F. Petit-Bertron, R. Bricard, M. Levasseur, A. Ramadan, 32. Hsu, C. L., and P. J. Bryce. 2012. Inducible IL-33 expression by mast cells is J. P. Girard, A. Herbelin, and M. Dy. 2009. IL-33 activates unprimed murine basophils directly in vitro and induces their in vivo expansion indirectly by regulated by a calcium-dependent pathway. J. Immunol. 189: 3421–3429. promoting hematopoietic growth factor production. J. Immunol. 183: 3591– 33. Su, Z., J. Lin, F. Lu, X. Zhang, L. Zhang, N. B. Gandhi, C. S. de Paiva, 3597. S. C. Pflugfelder, and D. Q. Li. 2013. Potential autocrine regulation of 26. Gonzalo, J. A., Y. Qiu, J. M. Lora, A. Al-Garawi, J. L. Villeval, J. A. Boyce, -33/ST2 signaling of dendritic cells in allergic inflammation. Mucosal Downloaded from C. Martinez-A, G. Marquez, I. Goya, Q. Hamid, et al. 2007. Coordinated in- Immunol. 6: 921–930. http://www.jimmunol.org/ by guest on September 27, 2021 1 SUPPLEMENTAL INFORMATION 2 3 4 5 Title: Transcriptional heterogeneity of mast cells and basophils upon activation1 6 7 Krishan D. Chhiba,1 Chia-Lin Hsu,1 Sergejs Berdnikovs1 and Paul J. Bryce1* 8

9 2 Supplemental Figures

10 1 Supplemental Table

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1 This work was supported by NIH Grant R01AI105839 (to P.B.)

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23 24 Supplemental Figure 1. GSEA analysis of hallmark gene set in mast cells after activation.

25 Enriched gene lists in IgE/Ag-crosslinked mast cells (A), IL-33 stimulated mast cells (B), or

26 select shared gene lists enriched in both IgE/Ag-crosslinked (C) and IL-33 stimulated (D) mast

27 cells. Enriched gene lists in IgE/Ag-crosslinked basophils (E), and IL-33 stimulated basophils

28 (F).

29 30 31

2 32 33 Supplemental Figure 2. Microarray validation of select genes by real-time RT-PCR. BMMCs

34 or BMBs were primed with 1 µg/ml anti-OVA IgE overnight and stimulated by addition of 0.5

35 µg/ml OVA (white bars), 10 ng/mL IL-33, or vehicle control (black bars) for 4 hours.

36 In BMMCs, gene expression of mouse Ccl1, Tnfa, Ccl5, Egr2, Fxyd6, and Il6 was determined by

37 real-time RT-PCR (A). In BMBs gene expression of mouse Ccl1, Plat, Cxcl2, and Il3 were

38 quantified (B). n=3-5 from independent cell cultures, *=p<0.05, **=p<0.01, ****=p<0.001 by

39 ANOVA.

3 Venn Diagram (Gene Lists)

Baso IL33, Baso IgE, Baso IgE, MC IL33, Baso IL33, Baso IgE, MC IL33, MC IgE Baso IgE, MC IgE Baso IgE, MC IL33 Baso IL33, MC IL33 Baso IL33, Baso IgE MC IgE MC IL33 Baso IgE Baso IL33 Groups MC IL33, MC IgE MC IgE MC IL33

Total 2 14 7 27 23 13 8 11 65 46 227 11 Elements Tnf Bcl2a1b Tpbg Pdcd1lg2 Pik3ip1 Idi2 Hacd3 Plat Abat Cd69 Ndrg2 Ppm1k 2010005H15Rik GsA1 Papss2 Traf1 Rai14 NCbia Paox Tfrc Rsad2 Slc7a11 Ikbke Mrps6 Mdk Cnnm2 Ifit2 Mxd1 Hist1h2bj Hcar2 Ccl1 Il6 Mrvi1 Egr3 Ppfibp2 Pou2f2 Tph1 Gng2 Irf5 Cd74 Fry Socs3 Heatr1 IRh1 Phlda1 Il1b Cx3cl1 Tubb6 BaS Cd52 Aldh1a3 Tnfrsf12a Srxn1 Lta Gls2 Mmp12 Ptx3 G0s2 Gm973 Fas Gadd45b Zbtb32 Cxcl16 Milr1 Sucnr1 Egr1 Slc6a4 Serpina3f Osbpl5 Tmtc2 Tmem51 Rassf4 Cited4 Csf2 Gmpr Trmt61a Il13 Zc3h12a Clec4e St6galnac4 Ttc39a Hivep3 Ccnd2 Frat2 Hist1h2bf Dgat2 Ccl7 Il1a Pik3ap1 6030419C18Rik NCb2 Tnfrsf1b Cxcl2 Fxyd6 Fam3b Mgst3 Nipal3 Klc4 Acer2 Serpinb2 Ccl4 Ngfr Rln3 Asb2 NCbiz Icam1 Ramp2 Galm Hmga2-ps1 Bcl2a1c N4bp2l1 Gabpb1 Aldh3a1 Bcl2a1d Vmp1 Fcgr2b Pilra St3gal5 Cyhr1 Rel Itgb4 Vegfc Dyrk1b Tppp3 Gchfr Gimap5 Rap1gap2 C3 Ppfia3 Areg Rnf180 Lrp4 Rhov Pdgfa Kcnk5 Srm Cdk5r1 Egr2 Nfatc1 Il33 Gbp3 Pim2 Hsd17b7 Lbh Crip2 Hip1r Snai3 Abcc5 Cxcl1 Chst11 Hdc Slc6a19 Amigo2 Elmo1 Gp5 Trim25 Hba-a1 Il3 Mapkapk3 Ccl3 Espn Dot1l Stx6 4930539E08Rik Hpn Antxr2 Rgs1 Gar1 Cxcl10 Tnfrsf9 Tspan32 Marcksl1 Mgst2 Guca2a Ak2 Mst1r Irf4 AI593442 Fgd2 Serpinb6b Ccrn4l Plxnd1 Alas1 Oasl1 Soat2 Clec7a Gzmd Dusp4 Cobll1 Akna Atp8b1 Hist1h2bk Srgap3 Dcstamp Eno2 Plk3 Scd1 Hmox1 NCbie Ssbp2 Bcl2 Dgat1 Ffar2 Car2 Emp1 Ccl5 Pprc1 Ppan Acss2 Hist1h2bm Lrrc36 Errfi1 Hvcn1 Mylk Retnla Sipa1l1 Aqp8 Serpina3g Agap1 Nxpe4 Col18a1 Tnfaip2 Ifit3 Chn2 Id1 Etv5 Lpcat2 Scimp Cd53 Relb Lrmp Hbegf Ckb Slc6a9 Pigz Rgs16 Asphd2 Ehd1 Fam117a Samhd1 P2rx5 Atad3a 1700012B09Rik Pcyt1b Spp1 Rims3 Kiz Ttc27 Bcl6 Hspa1a Il15 Cinp Fbxl21 Hist1h2bc Clec5a Iqgap2 1810009J06Rik Nphs2 Cdr2 Emc9 Cers6 Slc7a2 Rfx2 Iars Nabp1 6720475J19Rik C1ql2 Sdad1 Slco4a1 Eef1e1 Rnase2a Ets1 Crabp2 Pkib Wdr45 Tmem71 Ccl2 Pnpla7 Zfp467 Itga5 B3galt6 Hemt1 H2-M2 Trib3 Cx3cr1 Calca Ctps Il4i1 Fam160a2 Rrp15 Ceacam2 Deptor Hist1h2bn Ccl17 Amigo3 Oaz2 Smarca1 Fez1 Edn1 Nop58 Ypel3 Penk C5ar2 Bcl3 Il2ra Ccl24 Gyk Arg1 Fads3 Klf2 Gart Pdzk1ip1 Hspa1b Tpst2 Slco2b1 Rrp9 Endod1 Uchl1 Nxn Cd72 Slc6a13 Cdk19 Hbp1 Cyp51 Itpr3 Itk Gpr84 Rgcc Tmem86a Zhx2 Ch25h Timp1 Trp53inp1 Mdfic Zbx3 Cd83 6430571L13Rik Rrp12 Carhsp1 Fam69c Il2 Rhod Rgs2 Rilp Tpsab1 Ier3 Gbp2 Jakmip1 Abcb1b Cd82 Aldh1a2 Ada Mrps18b Hipk2 Ticam2 Sphk1 Ugcg Ahnak Itgb6 Ephx2 Clec4n Plk2 Tpst1 Adamtsl4 Atp10a Hist1h3f Ttc19 Bcat1 E330037I15Rik Vash1 Atp2a2 Oaf Slc46a3 Tpm3 Atxn1 Axl Bcl2l1 Il4 Slc37a2 Fam65b Il1rn SSa1 Tnfsf14 Car4 Grwd1 Cd34 Gdf15 BaS3 Fcrla Card11 Auts2 Lpl Wdr74 Cryba4 Syn2 Wdr75 Hist1h2be Bdh2 Pecam1 Flt1 Irgm2 Ifi27l2a Mmd Dusp2 Tnfrsf22 Slc6a12 Chst8 Sla Osm Avpi1 Dbp Anxa1 Lypd6 Cmpk2 Aldh18a1 Hist1h2bh Gpcpd1 Adora2b Lmo4 Rnf125 Xcl1 Rnf167 Rps6ka2 Tlr2 Gpr68 Gm5483 Cdc42ep5 Sgk1 Vasn S100a9 Sema7a Mmp13 Csrp3 Ccdc184 Ampd3 Ppif Tsc22d3 Fos Aqp3 Socs1 Bcl2l11 Pik3cd Pxmp2 Ptprf Tnfrsf4 Clec4b1 Vkorc1l1 Cmtm6 Inhba Id2 Mcf2l Cd300a Lif NCbid 40 41

42 Supplemental Table 1. List of gene identities common or unique to each cell type and

43 stimulation condition.

44

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1