Identification of the Cytokines, Growth Factors and Their Receptors Expressed by Human Blood Eosinophils: A Transcriptomics Approach

Eosinophils are mainly known to be produced in the bone marrow and in a type-2 (T2) immune environment, in vivo, in many prevalent diseases (e.g., allergies, asthma, atopic dermatitis, chronic sinusitis) as well as other high-burden diseases (e.g., eosinophilic esophagitis, gastritis, enteritis, colitis, life-threatening hypereosinophilic syndromes). Eosinophils are known to have negative effector functions on their surroundings via the release of four granule-stored toxic proteins, MBP (eosinophil major basic protein; PRG2), ECP (eosinophil cationic protein; RNASE3), EDN (eosinophil-derived neurotoxin; RNASE2) and EPX (eosinophil peroxidase). In addition to these toxic proteins, eosinophils release many cytokines and growth factors [1-3], although the list of mediators (i.e., cytokines and growth factors) produced by human mature eosinophils remains largely uncertain. In the present study, using several transcriptomic data sets, we identified what human eosinophils express as cytokines, growth factors, and receptors for such mediators. This analysis allowed us to identify: (1) mediators that eosinophils express for other types of cells and whose protein would act only in a paracrine way (i.e., eosinophils do not express their specific receptors), (2) mediators originating either from eosinophils themselves or exogenously from other cell types that can trigger intracellular signalling in eosinophils (i.e., eosinophils express their complete receptors), and (3) cytokines that eosinophils can bind via their expression of a specific subunit receptor but for which eosinophils lack a second subunit to form a complete receptor. At the University of Lille, blood eosinophils from four healthy donors were isolated from whole blood using MACSxpress Whole Blood human Eosinophil Isolation Kit (Miltenyi Biotech) as well as from participants with uncontrolled severe asthma (n = 21) or patients with refractory severe chronic sinusitis with nasal polyps (CRSwNP) (n = 18) using the EasySep Human Eosinophil Isolation Kit (STEMCELL Technology) as previously described [4]. At the University of Wisconsin-Madison, circulating blood eosinophils were obtained from three allergic donors, and were also purified by negative selection as previously described [5]. Rare visible contaminant cells were typically neutrophils or lymphocytes. Eosinophils were cultured with or without IL-3, IL-5 and GM-CSF for 4, 6 and 24 hours. Detailed RNA-sequencing in Lille and Madison, and depositions in public databases are provided in the Method sections in [4, 5]. Altogether, we found a total of 24 genes known to be genes coding for proteins that are either cytokines or growth factors according to www.uniprot.org and functional annotation in DAVID tools. Among the 24 genes, the expression level of IL16, IL1B, INHBA, OSM, TNFSF13B (BAFF) and VEGFA increased in isolated blood eosinophils from patients with uncontrolled severe asthma compared to healthy individuals, while the expression of IL16, IL18, INHBA, TNFSF13B and TNFSF14 increased in refractory CRSwNP. Thus, further investigations focused on the role of these mediators released by eosinophils in eosinophilic severe asthma, and/or CRSwNP might be of interest. In addition, we found a total of 37 genes expressed and coding for subunits of receptors for cytokines and growth factors. According to the expression of all subunits necessary to form complete and functional receptors, we identified that eosinophils should then be able to respond by intracellular signalling to 19 cytokines/growth factors, among which seven are expressed by eosinophils themselves to be able to act in an autocrine way (GRN, IL-16, IL-18, TGF-ß1, TRAIL, TWEAK and LIGHT; Figure 1A), while 12 mediators can be provided exogenously by other cell types (Figure 1B). Eosinophils also expressed one specific receptor subunit to bind IL-11, IL-12, IL-15, IL-17A, IL-17F, IL-2, IL-25 and IL-6, but lack another receptor subunit required for intracellular signalling in eosinophils by these eight cytokines (Figure 1C), suggesting that eosinophils may consume or trans-present some of these cytokines. Yet, as a limitation to this transcriptomics analysis, whether the lack of expression of a receptor subunit is necessarily accompanied by the absence of intracellular signalling in response to these ligands remains uncertain. Finally, 13 cytokines and growth factors expressed by eosinophils lack a specific receptor on eosinophils (Figure 1D), making these mediators available for surrounding cells, thus acting in a paracrine manner. Among these 13 genes, the expressions of IL1B, MYDGF, OSM, TGFA, and VEGFA were upregulated by IL-3 alone and the combination of the three beta-chain binding proteins (IL-3, IL-5 and GM-CSF). In conclusion, the present transcriptomics analysis allowed us to generate a list of cytokines and growth factors generated by eosinophils and available to act on surrounding cells to affect eosinophilic diseases. This short list includes the IL-1 s, which we have previously reported to be released from blood eosinophils, leading to activation of memory Th-17 lymphocytes and stimulation of fibroblasts to produce IL-6 and IL-8 in co-culture experiments [6-8]. Oncostatin-M, also part of this list, was very recently described as produced by human mature eosinophils and associated with uncontrolled severe asthma [4], while we also previously found that TNF-α plus IL-3-activated blood eosinophils could release high amounts of activin-A (INHBA) protein [9]. In addition, we identified other cytokines not expressed by eosinophils that should be possibly captured from the environment via a specific receptor subunit on eosinophils. Further analyses are necessary to demonstrate (1) that the identified genes produce proteins and (2) how the mediators released from or captured by eosinophils can affect other types of cells such as fibroblasts, endothelial cells, epithelial cells, as well as other immune cells. All authors have made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; and have been involved in drafting the manuscript or revising it critically for important intellectual content; and have given final approval of the version to be published. Each author has participated sufficiently in the work to take public responsibility for appropriate portions of the content, and they agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. We thank the research participants from whom the data were acquired for their contributions to this work. The research nurse coordinators in the UW-Madison Clinical Core facility (P.I. Loren Denlinger) for subject recruitment and screening, and Paul Fichtinger for the preparation of blood eosinophils (P.I. Sameer Mathur). We thank Jean-Pascal Meneboo, Frédéric Leprêtre, Shéhérazade Sebda, Delphine Beury, and Martin Figeac from the GO@L core genomics (PLBS UAR 2014—US 41) for their implication in generating transcriptomics data and first processing steps of RNA-sequencing analysis. This study was conducted according to the guidelines of the Declaration of Helsinki and approved by the University of Wisconsin-Madison (UW-Madison) Health Sciences Institutional Review Board (IRB#2013–1570, IRB#1999–292). At the University of Lille, the study was approved by the Institutional Review Board Sud-Est I (IRB# 2019-A01457-50) and was declared to the National Commission on Informatics and Liberty. At both sites, all participants provided written consent prior to participation. N. N. Jarjour received research funding from AstraZeneca for the extension of the National Institutes of Health-funded severe asthma research programme; and received consulting fees over the past 3 years from GlaxoSmithKline, Chiesi, and AstraZeneca related to asthma treatment. Delphine Staumont-Sallé received funding from AstraZeneca for a research study in DRESS syndrome; was investigator for clinical trials sponsored by AbbVie, Almirall, Amgen, AstraZeneca, Eli Lilly, Galderma, Leo Pharma, Novartis, Pfizer, Sanofi-Regeneron; received consulting fees from AbbVie, Almirall, AstraZeneca, Eli Lilly, Galderma, GSK, Leo Pharma, Novartis, Pfizer, Sanofi-Regeneron, UCB. Frédéric Dezoteux was investigator for clinical trials sponsored by AbbVie, Almirall, Amgen, AstraZeneca, Eli Lilly, Galderma, Leo Pharma, Novartis, Pfizer, Sanofi-Regeneron; received consulting fees from AbbVie, Almirall, Celltrion, Eli Lilly, Galderma, Leo Pharma, Novartis, Pfizer, Sanofi-Regeneron, UCB. These relationships with pharmaceutical companies are not relevant to the current study. Cécile Chenivesse declares research grants from AstraZeneca, GlaxoSmithKline, Santelys, and Novartis, personal fees from ALK-Abello, AstraZeneca, Boehringer-Ingelheim, Chiesi, Sanofi, and GlaxoSmithKline, and congress support from AstraZeneca, Boehringer Ingelheim, Chiesi, and Novartis. Guillaume Lefèvre received consulting fees, personal fees for advisory boards or meetings, and research funding from AstraZeneca and GSK. These relationships with pharmaceutical companies are not relevant to the current study. Mats W. Johansson received funding from Hoffmann-La Roche, outside of the submitted work. The rest of the authors declare that they have no relevant conflicts of interest. All the data used in this manuscript are available to the public. Raw data of RNA sequencing was deposited to SRA with assigned accession number (PRJNA1136662) and to the NCBI Gene Expression Omnibus (GEO) database and assigned accession number (GSE159782).