IL-33 binds to its receptor to trigger the activation of nuclear factor-kappa B (NF-B) and mitogen-activated protein kinases (MAPK) including c-Jun amino-terminal kinases (JNK), p38 MAPK (p38), and extracellular signal-regulated kinases (ERK). IL-33, which is a member of the IL-1 cytokine family [9], [10]. IL-33 binds to its receptor to trigger the activation of nuclear factor-kappa B (NF-B) and mitogen-activated protein kinases (MAPK) including c-Jun amino-terminal kinases (JNK), p38 MAPK (p38), and extracellular signal-regulated kinases (ERK). The activation results in production of Th2-associated cytokines including IL-5 and IL-13, which induce eosinophil maturation and mucus production, respectively. In a mouse model, airway inflammation was induced by administration of IL-33 [9], [11]. In addition, transgenic mice overexpressing IL-33 have been shown to exhibit airway inflammation [12]. These results suggest that the IL-33/ST2L axis is usually associated with the development of airway inflammation. Type 2 innate lymphoid cells (ILC2), originally referred to as natural helper (NH) cells, were discovered as a novel target of IL-33 [13]. Recent studies reported that lung ILC2 participated in the induction of airway inflammation in influenza virus-infected mice and papain-administrated mice [14], [15]. These studies indicate that lung ILC2 are a possible therapeutic target for airway inflammation. However, unfavorable regulators and effective inhibitors controlling ILC2 function have not yet been identified. We previously reported that soluble ST2 bound to IL-33 directly and inhibited its VE-821 binding activity for the IL-33 receptor by using cells of the murine thymoma cell line EL-4 that were stably transfected with ST2L (ST2L/EL-4 cells) [16]. However, it has not been determined VE-821 whether soluble ST2 affects natural IL-33-target cells such as ILC2. Here, we VE-821 isolated lung ILC2 from na?ve BALB/c mice and examined the responses of ILC2 to stimulation with IL-33. We also demonstrated that soluble ST2 suppressed the IL-33-mediated responses of lung ILC2. 2.?Materials and methods 2.1. Mice BALB/c mice were purchased from Japan SLC Inc. (Shizuoka, Japan) and housed in the animal research facility of Jichi Medical University. All experiments VE-821 were approved by the Animal Research Ethics Board of Jichi Medical University. 2.2. Antibodies and reagents Monoclonal antibodies against CD16/CD32, Alexa Fluor 700-conjugated CD45.2, fluorescein isothiocyanate-conjugated lineage cocktail (CD3, Gr-1, CD11b, CD45R, and Ter119), CD19, and CD49b, phycoerythrin (PE)-conjugated CD127 and IL-5, peridinin chlorophyll protein (PerCP)-Cy5.5-conjugated CD25, allophycocyanin-conjugated c-Kit and IL-4, PE-Cy7-conjugated Sca-1 were purchased from BioLegend. Streptavidin-conjugated brilliant violet (BV) 421, carboxyfluorescein diacetate succinimidyl ester (CFSE), zombie NIR dye, and brefeldin-A were also purchased from BioLegend. Monoclonal antibodies against biotinylated T1/ST2 and PerCP-eFluor710-conjugated IL-13 were purchased from MD Bioproducts and eBioscience, respectively. Propidium iodide (PI) was purchased from BD Biosciences. Murine IL-2 and IL-33 were purchased from PeproTech. 2.3. Cell culture Human embryonic kidney 293T (HEK293T) cells were cultured in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich) containing 10% fetal bovine serum (FBS). ST2L/EL-4 cells were cultured in RPMI 1640 medium (Sigma-Aldrich) containing 5% FBS, 50?M 2-mercaptoethanol (2-ME), and 6?g/ml blasticidin [16]. Lung ILC2 were cultured in RPMI1640 medium containing 10% FBS, 50?M 2-ME, and IL-2 Rabbit Polyclonal to DRD4 (20?ng/ml). 2.4. Purification of recombinant soluble ST2 Recombinant murine soluble ST2 tagged with V5 and His (ST2-V5) was transiently expressed in HEK293T cells and purified from serum-free culture supernatants as described previously [17]. VE-821 Deglycosylation of ST2-V5 with other groups). 4.?Discussion Here we showed that soluble ST2 suppressed the effect of IL-33 on lung ILC2 derived from na?ve BALB/c mice. IL-33 induced morphological change of na?ve lung ILC2 dramatically. IL-33-stimulated lung ILC2 exhibited augmented proliferation and upregulated expression of cytokine receptors and Th2-associated cytokines. Pretreatment with soluble ST2 significantly suppressed IL-33-induced responses. We demonstrated that a five-molar excess of soluble ST2 competed away the IL-33-mediated responses in ST2L/EL-4 cells and lung ILC2. Structural analysis using nuclear magnetic resonance (NMR) showed that the ternary complex of IL-33/ST2L/IL-1RAcP formed a stoichiometry of 1 1:1:1 [20]. The NMR-based study also proposed a complex formation model, in which IL-33 bound to ST2L first and then the IL-33/ST2L complex recruited IL-1RAcP. Soluble ST2 corresponds to the extracellular portion of ST2L, including the IL-33-binding site [1], [2]. Based on the structural analysis, it was predicted that equal amount of soluble ST2 and IL-33 formed into complexes. However, a sufficient suppressive effect was achieved when the ST2-V5/IL-33 molar ratio was.