Elsevier

Cytokine

Volume 136, December 2020, 155268
Cytokine

Interleukin 3-induced GITR promotes the activation of human basophils

https://doi.org/10.1016/j.cyto.2020.155268Get rights and content

Highlights

  • IL-3 induced 323 genes encoding surface proteins on human basophils.

  • GITR was preferentially induced on basophils by IL-3.

  • GITR promoted secretion of histamine, IL-4 and IL-13 by IL-3-activated basophils.

Abstract

Human basophils regulate allergic reactions by secreting histamine, interleukin 4 (IL-4) and IL-13 through key surface receptors FcεRI as well as IL-3R, which are constitutively expressed on basophils. IL-3/IL-3R signaling axis plays key roles in regulating the development and activation of basophils. We and others have shown that IL-3-induced surface receptors e.g. ST2, IL-17RB and IL-2 receptors regulate the biology of basophils. However, the expression and function of IL-3-induced surface proteins on human basophils remain to be elucidated. We in this study aimed to identify new basophil activation regulators by transcriptomic analysis of IL-3-stimulated basophils. Gene expression microarray analysis of IL-3-treated basophils revealed 2050 differentially expressed genes, of which 323 genes encoded surface proteins including GITR. We identified that GITR was preferentially induced by IL-3 rather than anti-IgE, IL-33, fMLP and C5a. IL-3-induced GITR was suppressed by inhibitors targeting JAK2, PI3K and MEK1/2. Stimulation of IL-3-treated basophils by GITR enhanced the expression of IL-4 and IL-13. Moreover, IgE-mediated degranulation was enhanced by GITRL in the presence of IL-3. This transcriptomic analysis of IL-3-activated basophils helps to identify novel activation regulator. IL-3-induced GITR promoted the activation of basophils, adding new evidence supporting GITR as an important player in Th2-associated immune responses.

Introduction

Basophils are important effector cells contributing to allergic reactions by secreting histamine and LTC4 that are prestored in granules as well as effector cytokines IL-4 and IL-13 [1], [2], [3], [4]. This is regulated by key receptors FcεRI as well as IL-3R, which are constitutively expressed by human basophils. IL-3R/IL-3 signaling axis is crucial for both of the activation and development of basophils [3], [5], [6]. We and others have found that activated basophils by IL-3 express multiple surface receptors and mediators e.g. ST2, IL-17RB, IL-2RA and IL-3, which promote the activation of basophils [7], [8], [9], [10], [11], [12]. This indicates that the function of basophils is co-regulated by constitutively expressed and induced surface receptors. Characterization of IL-3-activated basophils may help to identify new regulators of human basophils.

Transcriptomic analysis has been widely used to decipher global gene expression changes between groups. Through gene expression microarray analysis of IL-3-stimulated basophils, we revealed multiple differentially expressed genes including GITR. GITR is a member of the TNF receptor family which is known to be expressed by T cells, NK cells and type 2 innate lymphoid cells (ILC2) [13], [14], [15]. A number of studies have implicated that GITR regulates allergic reactions [14], [16], [17], [18]. Activation of GITR exacerbates allergic airways inflammation with enhanced expression of Th2 cytokines and IgE [16]. In addition, activation of GITR on human and murine ILC2 promotes the expression of IL-5, IL-9 and IL-13 and allergic lung inflammation [14]. Through gene expression microarray analysis, we have previously identified that GITR is highly expressed in allergen challenged PBMCs from allergic patients [19], [20]. However, whether GITR is expressed on human basophils are not known yet.

In this study, we reported that GITR was preferentially induced by IL-3. We identified that IL-3-induced GITR on human basophils was dependent on JAK2, PI3K and MEK1/2 pathways. Stimulation of GITR by GITRL enhanced the activation and degranulation of IL-3-primed basophils.

Section snippets

Ethics statement

The usage of buffy coats from whole blood of healthy donors was approved by the ethics committee of Xuzhou Medical University and Xuzhou Red Cross Blood Center. The written consent was obtained from blood donors according to instructions by the ethics committee of Xuzhou Red Cross Blood Center.

Reagents and antibodies

Human CRTH2 microBead kit and Basophil isolation kit were obtained from Miltenyi (Germany). Anti-human CD4-FITC (OKT4), anti-human CRTH2-PE (BM16), anti-human CD123-FITC (6H6), anti-human HLA-DR-Pacific

IL-3 induced thousands of genes changed significantly in human basophils

IL-3/IL-3R signaling axis plays key roles in the maturation, survival and activation of basophils. Characterization of IL-3-induced genes may help to identify new regulators of human basophils. Hence, we performed gene expression microarray analysis of flow cytometric sorted basophils (SSClowCD4 CRTH2+) with or without IL-3 treatment [10], [21], [22], [23]. This led to identification of 2050 transcripts that were differentially expressed (DE) (Table S1). These DE genes included a list of genes

Discussion

IL-3 acts as a master regulator of basophils, which play indispensable roles in allergic immune responses. Multiple co-regulators of human basophils have been identified to be induced by IL-3. In this study, using gene expression microarray analysis we identified a large body of surface protein-encoding genes that were induced by IL-3. We for the first time showed that GITR was preferentially induced by IL-3 via different pathways. We further demonstrated that activation of GITR promoted the

Funding

This work was supported by the National Natural Science Foundation of China (grant no. 31800750), the Youth Innovation Team Grant and the Starting Grant by Xuzhou Medical University (grant no. D2018009).

CRediT authorship contribution statement

Li Hong: Investigation. Yangyang Tang: Investigation. Shuai Pan: Investigation. Meizhen Xu: Investigation. Shi Yanbiao: Investigation. Sijia Gao: Investigation. Chao Sui: Investigation. Cheng He: Investigation. KuiYang Zheng: Writing - review & editing. Renxian Tang: Writing - review & editing. Zhixu Shi: Writing - review & editing. Qingling Wang: Supervision, Writing - review & editing. Hui Wang: Investigation, Formal analysis, Supervision, Writing - original draft, Writing - review & editing,

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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