Notch signaling pathway regulates the growth and the expression of inflammatory cytokines in mouse basophils

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Research paper

Notch signaling pathway regulates the growth and the expression of inflammatory cytokines in mouse basophils ⁎

Shuo-yao Qua,1, Jia-Ji Linb,1, Jian Zhanga, , Li-Qiang Songa, Xue-Min Yanga, Chang-gui Wua, a b

⁎

Department of Pulmonary and Critical Care Medicine, Xijing Hospital, Fourth Military Medical University, China Department of Neurology, Tangdu Hospital, Fourth Military Medical University, China

A R T I C L E I N F O

A B S T R A C T

Keywords: Notch Basophil Immune regulation MAPK

Basophils (BAs) are the least common granulocytes of all leukocytes, but they play an important role in orchestrating of chronic allergic inflammation. The Notch signaling pathway is a highly conserved pathway that influences cell lineage decisions and differentiation during various stages of development. However, the relationship between Notch signaling and BA remains to be elucidate. Here, we report that several Notch signaling molecules were found to be expressed in BAs. γ-secretase inhibitor (GSI) treatment increase BAs apoptosis, and suppress BAs proliferation. Furthermore, GSI reduced BAs in the S phase, with a concomitant accumulation in G1 and G2 phases. In addition, GSI also significantly down-regulated mRNA levels of cytokines IL-4, IL-6 and IL-13 induced by A23187, and this effect was dependent on MAPK pathway. Finally, IL-6 inhibition was specifically associated with ERK and IL-13 with JNK. Therefore, Notch signaling regulates BA biological function, at least partially via the modulation of MAPK.

1. Introduction Basophils (BAs) are a rare type of granulocyte that matures in the bone marrow and are subsequently released into the peripheral blood. In recent years, BAs have been gaining attention as critical to the pathogenesis of allergic inflammation, and many studies have demonstrated that BAs play a pivotal role in inflammatory diseases, such as allergic asthma. Dendritic cells (DC), for example, have been found to be dispensable for this disease process, whereas antigen presentation by BAs was necessary and sufficient for allergen-induced activation of Th2 responses in vitro and in vivo [1]. Recent studies have also revealed that BAs perform not only as antigen presenting cells (APCs) but also as effector cells that preferentially induce Th2 cells in response to complexes of antigen plus antigen specific IgE [2]. Furthermore, BAs have been considered as ‘innate type 2 cells’, which could lead the development of adaptive type 2 immunity by providing the initial IL-4 used by CD4+ T cells in their differentiation to the Th2 phenotype [3]. The Notch signaling pathway is an important pathway that regulates development with high conservation across species [4]. In the hematopoietic tissues, Notch has been shown to influence cell lineage decisions and differentiation during various stages of development [5].

Specifically, Notch receptors and ligands are involved in the interaction between APCs and T cells. Furthermore, it has been demonstrated that several Notch signaling ligands are expressed on Mast cells [6], and that these molecules are involved in their proliferation [7,8]. BAs share several characteristics with tissue-resident Mast cells, including surface expression of the high-affinity IgE receptor (FcεRI) and the release of allergy-related chemical mediators such as histamine in response to various stimuli [9,10]. Th2 cytokines have been implicated in the pathogenesis of allergic asthma. IL-4, for example, plays a vital role in driving the differentiation of naive T cells into Th2 cells [11], and IL-13 induces eosinophils and Th2-recuiting chemokines. Transcriptional activation of cytokines in various types of APCs can be the result of induction by mitogenactivated protein kinases (MAPKs) [13]. MAPKs activated by various environmental stimuli, in fact, regulate the transcriptional activity of many genes involved in maintaining cellular homeostasis. There are three major groups of MAPKs in mammalian cells that are important regulatory proteins that transduce various extracellular signals into intracellular events: the extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK)/stress-activated protein kinase, and p38 subfamilies [12].

Abbreviations: APC, antigen presenting cells; BA, Basophil; CCK-8, cell counting kit-8; DC, dendritic cell; ERK, the extracellular signal-regulated protein kinase; GSI, γ-secretase inhibitor; IL, interleukin; JNK, c-Jun N-terminal kinase; MAPKs, mitogen-activated protein kinases; TGF-β, transforming growth factor-β; PBS, phosphate-buffered saline ⁎ Corresponding authors at: Department of Pulmonary and Critical Care Medicine, Xijing Hospital, Fourth Military Medical University, No. 127, Changle Western Road, Xi'an, Shanxi 710032, China. E-mail address: [email protected] (C.-g. Wu). 1 These authors contributed equally to this study. http://dx.doi.org/10.1016/j.cellimm.2017.05.005 Received 26 November 2015; Received in revised form 8 May 2017; Accepted 25 May 2017 0008-8749/ © 2017 Elsevier Inc. All rights reserved.

Please cite this article as: Qu, S.-y., Cellular Immunology (2017), http://dx.doi.org/10.1016/j.cellimm.2017.05.005

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2.5. FACS analysis

Whether Notch signaling and/or MAPKs regulate the antigen presentation function of BAs remains unclear. Here, expression of Notch signaling molecules was investigated in primary cultured mouse BAs. Furthermore, as these molecules may be involved in regulating the production of inflammatory mediators in asthma, a Notch inhibitor, γsecretase inhibitor (GSI), was further evaluated as a potential therapeutic modality for the prevention of the onset and severity of allergic asthma. Our results demonstrated that GSI significantly inhibited increased production of inflammatory cytokines IL-6 and IL-13 induced in BAs, and that their expression was mediated through the modulation of MAPKs, JNK and ERK, respectively. Therefore, Notch signaling might play a central role in induction of allergic airway inflammation mediated BAs.

For cell cycle analysis, cells (1 × 106) were fixed in 75% alcohol for 30 min at 4 °C, rinsed with cold PBS three times, and incubated at 37 °C for 30 min in 1 mL of PBS containing 40 μg propidium iodide (PI; Sigma; St. Louis, MO, USA) and 100 μg RNase A (Sigma). For apoptosis analysis, was detected with the Annexin V-FITC Apoptosis Detection Kit I (Pharmingen, San Diego, CA, USA), according to the manufacturer’s instructions. Cell suspensions were filtered through a nylon membrane and resuspended in FACS buffer for flow cytometry (PBS, 2% FCS, 0.05% NaN3). All samples were analyzed for fluorescence staining and DNA content on the FACScalibur™ (BD Immunocytometry Systems; San Jose, CA, USA). 2.6. ELISA assays

2. Materials and methods BAs (1 × 106 cells/mL) were seeded into 6-well plates. Supernatants from treated cells were screened for IL-4, IL-6, and IL-13 protein levels with a human ELISA kit according to the manufacturer's instructions (ELISA kits; Neobioscience; ShenZhen, China).

2.1. Cells Mice’s bone morrow was flushed, and a single-cell suspension was prepared, followed by erythrolysis in ammonium chloride-potassium buffer. Bone marrow basophil (BMBA) were prepared by culturing BM cells in the presence of rIL-3 (300 pg/ml) for 1 week, followed by isolating the CD49b+ cells using the IMag system with biotinylated antiCD49b and streptavidin-conjugated magnetic particles (BD PharMingen, San Diego, CA, USA). FACS results demonstrated that the CD117−CD11c−CD49b+FcεRI+ cells were highly pure and viable basophils (Fig. S1).

2.7. Real-Time PCR Total RNA was extracted with TRIzol reagent (Life Technologies), and cDNA was synthesized with a reverse transcription kit (Toyobo; Osaka, Japan). Real-time PCR was performed with the SYBR Premix Ex Taq kit (Takara; Otsu, Japan) on the ABI PRISM 7300 (Applied Biosystems; Foster City, CA, USA). β-Actin was used as an internal control. Sequences of the primers used were the following: IL-4 Forward (F): 5′-ATGGGTCTCACCTCCCAACTGCT-3′, IL-4-Reverse (R): 5′-CGAACACTTTGAATATTTCTCTCTCCAACTGCT-3′; IL-6-F: 5′-ATGAACTCCTTCTCCACAAGCGC-3′, IL-6-R: 5′-GAAGAGCCCTCAGGCTGGACTG-3′; IL-13-F: 5′CCACGGTCATTGCTCTCACTTGCC-3′, IL-13-R: 5′-CCTTGTGCGGGCAGAATCCGCTCA-3′; β-actin-F: 5′-TAGTTGCGTTACACCCTTTCTTG-3′, βactin-R: 5′-TCACCTTCACCGTTCCAGTTT-3′.

2.2. RT-PCR RNA was extracted with TRIzol reagent (Life Technologies), and cDNA was synthesized with reverse transcriptase (Fermentas; Vilnius, Lithuania) according to the manufacturer’s instructions. RT-PCR was performed with the 2 × Taq Master Mix (KangWei, Beijing, China) on a thermocycler. Sequences of the primers used are listed in Supplementary Table S1. Amplified fragments were analyzed by electrophoresis on a 2% agarose gel.

3. Results 3.1. Expression of Notch-related genes in BAs We primary cultured BAs for 7 days and enriched them by FACS according to SSClowCD117−CD11c−CD49b+FcεRI+ (Fig. S1). Then, the expression of Notch related genes was examined in BAs. RT-PCR was performed with primers specifically targeting the mouse Notch1, Notch2, Notch3, Notch4, Jagged1 and Jagged2, Delta-like1, Deltalike3, Delta-like4 and Hes1 and 5, and β-actin as an internal control. The results revealed that Notch3, Notch4, and Delta-like 4 were highly expressed in BAs (Fig. 1A), while Notch2 was not detected. Two of the main Notch downstream genes, Hes1 and Hes5, were also highly expressed in BAs. Expression of Notch1, Notch3, Delta-like4, and Jagged2 in BAs was further confirmed by Western blot (Fig. 1B).

2.3. Cell proliferation assay Cells (103/well) were plated in 96-well plates in 200 μL of medium and incubated at 37 °C. Following treatment, reagent from the Cell Counting Kit-8 (CCK-8)10 μL (Dojindo) was added to wells and incubated for 2 h. The absorbance of the supernatant was measured at 450 nm. The cell viability curve was determined by plotting the mean value and standard deviation of the optical density. All experiments were done in 4 times.

2.4. Western blot analysis 3.2. Notch signaling inhibitor (GSI) suppresses the growth of BAs Cells were lysed in phospho-lysis buffer (50 mM Tris-Cl, pH7.4, 150 mM NaCl, 1 mM MgCl2, 0.5% NP-40, 1 mg/mL bovine serum albumin [BSA], and 0.1 mM PMSF), and protein concentrations were determined with the BCA Protein Assay reagent (Pierce; Thermo Scientific; Rockford, IL, USA). Samples were separated by SDS-10% polyacrylmide gel electrophoresis, followed by Western blotting. Blots were incubated with the following primary antibodies: anti-Notch1 (ab52627), anti-jag2 (ab-109627), pJNK123(ab59196), and JNK123 (ab124956; Abcam; Cambridge, MA, USA); anti-Notch3 (sc-5593), antidelta4 (sc-28915) and phospho-erk (sc16982; Santa Cruz Biotechnology; Dallas, TX, USA); p38 (#4511), p38 (#9212), erk (#9102; Cell Signaling Technology; Boston, MA, USA). anti-rabbit-immunoglobulin G was used as secondary antibody.

In order to investigate the role of Notch signaling in BAs growth, cells were exposed to an inhibitor of Notch signaling, GSI. The growth of BAs exposed to GSI, a γ-secretase inhibitor IX (Calbiochem, La Jolla, CA), which was used at the concentration of 75 μM/L, with DMSO as a control. was significantly decreased relative to control cells (p < 0.05, Fig. 2A). These data indicated that inhibition of Notch signaling suppressed the growth of mouse BAs in vitro. 3.3. GSI induces apoptosis and result in cell cycle arrest at the G1/G2 phase Cell cycle parameters and apoptosis were examined by flow cytometry in order to identify mechanisms underlying the inhibition of BA 2

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Fig. 1. Notch signaling molecules are expressed by BAs. Expression of Notch signaling molecules in BA as determined by RT-PCR in (A) and by Western blot in (B). βactin was used as an internal control for both RT-PCR and Western blots. Primary antibodies, Notch1, Notch3, Deltalike 4, and Jagged-2, are indicated.

The levels of the cytokines were compared in A23187 and A23187 + GSI treated BAs. Expression levels were first determined based on RNA. The mRNA levels of IL-4, IL-13, and IL-6 were significantly less than in control cells (p < 0.05; Fig. 3A). Subsequently, the supernatants of cells were collected to detect the levels of these proteins secreted by BAs. Interestingly, IL-6 and IL-13 concentrations were influenced by the Notch inhibitor GSI, whereas changes in IL-4 were not detectable by ELISA. (Fig. 3B). These results indicated that Notch signaling might play an important role in the regulation of the secretion of cytokines by BAs.

growth. The results demonstrated that GSI treatment induced apoptosis 0.44% (control) vs 5.9% (24 h) vs 29.86% (48 h) (p < 0.05, Fig. 2B), in a time-dependent manner. Interestingly, inhibition of Notch decreased the percentage of cells in S phase, while decreasing the number of cells in the G1 and G2 phases of the cell cycle (Fig. 2C). Both responses were time-dependent.

3.4. Notch signaling regulates cytokine secretion by BAs ELISA was performed in order to detect the secretion of Th2 cytokines by BAs. A23187, which is a calcium ionophore, significantly induces secretion of Th2 cytokines, such as IL-4 and IL-13, in BAs. Therefore, the influence of Notch signaling on cytokine secretion of BAs in response to A23187 was examined by combining treatment with GSI.

3.5. GSI inhibits the activation of JNK and ERK Activation of MAPKs is associated with the expression of pro-

Fig. 2. Notch inhibitor, γ-secretase, inhibits BA growth through apoptosis and G1/G2 phase arrest. (A) Proliferation of BA under the treatments indicated was determined with the CCK8 assay. (B) and (C) Flow cytometric analysis was performed to detect apoptosis and cell cycle distribution in BA following treatment with GSI. Cells were stained with Annexin-V and PI after treatment with different concentrations of GSI for the indicated time periods. The result represent mean of four independent experiments. Bars represent mean ± S.D. (n = 4). (*: p < 0.05, **: p < 0.01 vs. SHAM group; △: p < 0.05, △△: p < 0.01, vs GSI 24 h group; #: p < 0.05, ##: p < 0.01, vs DMSO 24 h group; & : p < 0.05, & & : p < 0.01, vs GSI 48 h group).

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Fig. 3. Protein levels of inflammatory cytokines IL-5 and IL13 are altered by γ-secretase. The supernatants of different group cells were collected 48 h after treatment with GSI. (A). mRNA levels of IL-4, IL-5, and IL-13 were determined by real-time PCR; (B). The concentrations of IL-4, IL-5, and IL-13 in supernatants were determined by ELISA in .The result represent mean of four independent experiments. Bars represent mean ± S.D. (n = 4). (*: p < 0.05, **: p < 0.01 vs. SHAM group; #: p < 0.05, ##: p < 0.01, vs GSI 24 h group).

reported to promote naive T cells to differentiate into Th1, while the level of Jagged-2 on was elevated the surface of APCs in a pro-Th2/ Th17 environment. BAs share several characteristics with tissue-resident mast cells, and many studies have demonstrated that Notch signaling confers antigen-presenting cell functions on mast cells [19]. Based on these findings, Noch was investigated in this study as a pathway involved in the regulation of the biological function of BAs. Our results demonstrated that Notch signaling molecules Notch1, Notch3, and Delta-like 4 were highly expressed on BAs, as well as critical downstream genes, Hes1 and Hes5. The Notch inhibitor GSI interfered with the induction of the inflammatory response indicating that Notch may be involved in the activity of BAs in normal and disease processes. Our data demonstrated that inhibition of Notch signaling suppressed cell growth of BAs. GSI treatment specifically induced apoptosis, and this effect was time-dependent. Inhibition of Notch furthermore shortened S phase, and arrested cells in G1 and G2. Both effects were time-dependent. These results indicate that Notch signaling positively regulates the proliferation and survival of BAs. An increasing number of studies demonstrate that Notch signaling is related to the proliferation of leukemia cell lines. Notch signaling is a potent inducer of growth arrest and apoptosis in a wide range of B-cell malignancies [20]. The growth of two kinds of myeloid leukemia cell lines induced by Notch ligands was suppressed by Notch activation [21]. Interestingly, Notch signaling was found to regulate T cell proliferation induced by dendritic cells [22]. Th2 cytokines play a vital role in the establishment of the inflammation of allergic asthmatic airways. IL-4 is the key Th2 cytokine in allergic asthma, and it is involved in inducing differentiation of Th2 cells and suppressing the stimulation of inducible Tregs (iTreg). IL-5 and IL-13 underlie bronchial hyper-responsiveness, over-production of mucus, thickening of smooth muscle, and subepithelial fibrosis [23,24]. mRNA levels of IL-4, IL-13, and IL-6 in our experiments were significantly less than in controls. Interestingly, only IL-5 and IL-13 concentrations in cell supernatants were decreased. We speculated that the post transcriptional regulation mechanism may be involved in regulating IL-4 expression. These results were similar to Notch signaling function in other APCs [25]. Taken together, these results indicated that Notch signaling might play an important role in the regulation of the secretion of cytokines by BAs. The MAPK cascade is an important signaling pathway in

inflammatory cytokines in various cell types. To determine whether the inhibition of IL-6 and IL-13 expression by GSI was mediated through interference of MAPK signaling in BAs, the activation of MAPKs was examined in BAs treated with GSI. BAs were exposed to GSI (10 μmol/ mL) for 24 h followed by incubation with A23187 for 2 h. Cell lysates were analyzed by Western blot. The A23187-induced phosphorylation of JNKp54/p46- and ERKp44/p42 was reduced in BAs treated with GSI (p < 0.05 and p < 0.01 vs. control, respectively; Fig. 4A). However, phosphorylation changes in p38 were not detectable by Western blot. To further investigate the influence of JNK- and ERK-inhibition by GSI on expression of proinflammatory cytokines in BAs, cells were treated with pharmacological agents that inhibit JNK- and ERK, and mRNA levels of cytokines were examined by real-time RT-PCR. Treating BAs with the selective JNK inhibitor, SP600125 (50 μM), and the ERK inhibitor, U-0126 (10 μM), blocked A23187-induced IL-6 and IL-13 gene expression (Fig. 4B). These data demonstrated that the inhibition of IL-6 and IL-13 expression by the Notch inhibitor GSI, was mediated, at least in part, by the inhibition of A23187-induced activation of JNK and ERK pathways. 4. Discussion Th1/2 bias is an important feature of allergic asthma, and increases in Th2 cytokines are observed in in vivo asthmatic models [14]. Such cytokines may be the basis of many pathological symptoms of asthma. IL-4 is a key Th2 cytokine in the development of allergic asthma, as it is involved in inducing the differentiation of Th2 cells while suppressing the induction of Tregs [15]. Recently, BAs have become the focus of studies on Th2 cell differentiation, especially as the source of the initial burst of IL-4 and even as the antigen-presenting cell [16]. This initial release of IL-4 could induce naïve Th0 to differentiate into Th2 and if it were prevented, subsequent pathological events perhaps would be reduced to a minimum. Notch receptors and ligands have had a long established role in the interaction between many APC and T cells, but the downstream effects elicited by different Notch receptors and ligands vary among cell types [17]. The expression of intracellular Notch1 in CD4+ T cells resulted in Th1 differentiation. And this effect was associated with IL-12-independent expression of T-bet. Notch1 could be immunoprecipitated with Tbx21 chromatin regions that were isolated from a T-cell hybridoma [18]. Delta-like1 and Delta-like4, for example, have been 4

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Fig. 4. JNK and ERK mediate expression of inflammatory cytokines IL-6 and IL-13 in BAs. (A) BAs were treated with PBS, A23187, or A23187 + GSI for 72 h. Protein expression/phosphorylation was determined by Western blotting for p38, Erk, and JNK as indicated. β-actin was used as the loading control. The expression of active p38, Erk and JNK in different groups. Quantification of Western blotting was performed by densitometry analysis. Expression values were normalized to β-actin. Data were presented as mean ± SD (*p < 0.01, **p < 0.01 vs. sham; # p < 0.05 vs. A23). (B) ELISA to detect levels of IL-6 and IL-13 in the supernatants of BAs treated with the JNK inhibitor, SP600125 (50 μM), and the ERK inhibitor, U-0126 (10 μM). The result represent mean of four independent experiments. Bars represent mean ± S.D. (n = 4). (*: p < 0.05, **: p < 0.01 vs. A23187 + GSI group; △: p < 0.05, △△: p < 0.01, vs A23187 + U0126 group).

expression in BAs. These results may help to better understand the cellular basis of Notch-mediated inhibition of asthmatic responses, and may contribute to the development novel therapies for treatment of asthma.

inflammatory responses [26]. The signaling pathways include MAPKs p38, ERK, and JNK, which play a potential role in the regulation of inflammatory response [27]. Persistent activated MAPK signals are a consequence of positive or double negative feedback loops with the result of perpetuating a signal after removal of an external cell stimulus. ERK1/2 favors Th2 differentiation through multiple mechanisms, including priming of immune cells for the transcription of cytokines which plays an important role in the pathogenesis of asthma [28]. In contrast to ERK1, JNK favors Th1 differentiation and plays an important role in corticosteroid-resistant asthma. The use of JNK inhibitors has been shown to reduce the accumulation of inflammatory cells in BALF, as well as cytokine release, serum IgE production, and smooth muscle proliferation in acute and asthmatic animal models [29]. p38 MAPK inhibitors have also been shown to reduce the release of inflammatory antigens and allergic inflammatory cytokines in animal models [30]. Interestingly, cells isolated from the BALF of severe asthmatic patients became sensitized by the combination of the p38 MAPK inhibitor and dexamethasone [31]. In the present study, GSI specifically inhibited A23187 induced activation of JNKp54/p46- and ERKp44/p42 MAPKs and the production of IL-6 and IL-13 in BAs. In addition, JNK- and ERK-specific inhibitors, SP600125 and U-0126, were used to demonstrate that JNK and ERK, regulated IL-6 and to some degree, IL-13 expression in BAs, respectively. And it has been reported that Notch regulate JNK and ERK positively in many kinds of cells [32,33]. So, these data indicate that Notch signaling may promote IL-6 and IL-13 gene and protein expression through JNK- and ERK-MAPK pathway respectively. In summary, the Notch signaling pathway was found to play a positive role in the regulation of the proliferation, apoptosis, and cytokine

The role of each author in the study Shuo-Yao Qu: collection and/or assembly of data, data analysis and interpretation, manuscript writing; Jia-Ji Lin: collection and/or assembly of data; Jian Zhang: cell culturing and Western blot, collection and/or assembly of data; Li-Qiang Song: RT-PCR, collection and/or assembly of data. Xue-Min Yang: cell culturing, q-PCR, collection and/ or assembly of data; Chang-Gui Wu : conception and design, final approval of manuscript. Acknowledgments This work was supported by the National Natural Science Foundation of China – China (81200018). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cellimm.2017.05.005. References [1] C.L. Sokol, N.Q. Chu, S. Yu, S.A. Nish, T.M. Laufer, R. Medzhitov, Basophils function as antigen-presenting cells for an allergen-induced T helper type 2 response, Nat. Immunol. 10 (2009) 713–720.

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