YM-341619 suppresses the differentiation of spleen T cells into Th2 cells in vitro, eosinophilia, and airway hyperresponsiveness in rat allergic models

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Abstract

T helper (Th) 2 cells play a central role in the pathogenesis of allergic diseases such as allergic asthma, atopic dermatitis, and allergic rhinitis. We have found that YM-341619 hydrochloride, which suppressed IL-4-induced STAT6-dependent reporter gene expression, inhibited the differentiation of mouse spleen T cells into Th2 cells in vitro. YM-341619 suppressed the production of IL-4 and the expression of GATA-3 mRNA, a Th2 transcription factor, in T cells cultured with anti-CD3 antibody and anti-CD28 antibody in the presence of IL-4. In contrast, the production of IFN-γ and the expression of T-bet mRNA, a Th1 transcription factor, in T cells cultured with anti-CD3 antibody in the presence of IL-12, were not effected by YM-341619. Orally administered YM-341619 (0.003–0.03 mg/kg) reduced the plasma IgE level of DNP–Ascaris-sensitized rats, but not the IgG2a level. YM-341619 suppressed IL-4 and IL-13 production in the splenocytes of these DNP–Ascaris-sensitized rats without augmenting IFN-γ production. YM-341619 also dose-dependently suppressed eosinophil accumulation in the lung (0.003–3 mg/kg, p.o.) and airway hyperresponsiveness (0.3–3 mg/kg, p.o.) induced by repeated exposure to ovalbumin in ovalbumin-sensitized rats. These results suggest that YM-341619 has the ability to suppress allergen-induced Th2 responses by selectively inhibiting the differentiation of CD4+ T cells into the Th2 subset.

Introduction

Naive CD4+ T helper (Th) cells differentiate into two distinct subsets, Th2 and Th1, as determined by cytokine production profiles and functional ability (Mosmann et al., 1989). Th2 cells produce IL-4, IL-5, IL-10, and IL-13, and control humoral immunity against parasites and allergic reactions. Th1 cells produce IFN-γ and are involved in cell-mediated immunity against intracellular pathogens. Differentiation into Th2 or Th1 is induced by antigen stimulation of T cell receptors. Although these differentiation programs are influenced by the type of antigen, its concentration, and the ligation of costimulatory molecules, etc., the most important regulators are cytokines (Constant et al., 1997). IL-4 and IL-12 are the principle cytokines that drive naive CD4+ T cells to differentiate into Th2 and Th1, respectively.

Several of the transcription factors involved in differentiation into Th1/Th2 have been identified. Among these, signal transducer and activator of transcription (STAT) 6, which is an essential transcription factor for IL-4 and IL-13 signaling, is known to play an important role in Th2 cell differentiation because STAT6-deficient mice fail to develop Th2 cells (Nelms et al., 1999). GATA-3, which is induced by IL-4 through STAT6, has also been identified as a Th2 differentiation factor (Ouyang et al., 1998), and is known to be a Th2-type cell marker (Ray et al., 1999). GATA-3 establishes transcriptional competence for the Th2 cytokine gene cluster (IL-3/IL-4/IL-5) via chromatin remodeling (Lee et al., 2000). Although naive CD4+ T cells express a low level of GATA-3 mRNA, the expression is markedly up-regulated in developed Th2 cells (Zhang et al., 1997). In contrast, STAT4, which is an essential transcription factor for IL-12 signaling, is important for Th1 cell differentiation (Yu et al., 2007). T-bet also plays an important role in Th1 cell differentiation by promoting chromatin remodeling at the IFN-γ locus (Mullen et al., 2001), and is known to be a Th1-type cell marker (Szabo et al., 1995).

Allergic asthma is characterized by reversible airflow obstruction, chronic bronchial or pulmonary eosinophilia, an elevated plasma IgE level, and airway hyperresponsiveness (Bousquet et al., 1990). It is believed that excessive Th2-driven responses result in the development of allergic asthma (Epstein and Epstein, 2006). In allergic asthmatic patients, activated CD4+ Th2 cells have been identified in both the bronchoalveolar lavage (BAL) fluid and airway biopsies (Humbert et al., 1999, Robinson et al., 1992). In addition, the number of Th2 cells in the airway of patients has been shown to increase after antigen challenge (Robinson et al., 1992). Th2 cytokines such as IL-4, IL-5, and IL-13 produced in activated CD4+ Th2 cells mediate a series of events in the inflammatory cascade that lead to the development of allergic asthma, that is, IL-4 mediates B cell maturation and IgE isotype class switching; IL-4, IL-5, and IL-13 mediate the induction and maintenance of eosinophilia in the lung (Coffman et al., 1989, Nakajima et al., 1992); and IL-13 mediates mucus hypersecretion and airway hyperreactivity (Wills-Karp et al., 1998).

To find new agents that suppress the differentiation of naive CD4+ T cells into Th2 cells, our in-house chemical libraries were subjected to high throughput screening using the IL-4-induced STAT6-dependent reporter gene expression system. This screening resulted in the discovery of a novel compound, YM-341619 hydrochloride (Fig. 1), which suppressed the differentiation of T cells prepared from mice splenocytes into the Th2 subset, but not the Th1 subset. In this study, we investigated the pharmacological effect of YM-341619 both in vitro and in rat asthma models.

Section snippets

Animals

Female C57BL/6 mice (7–8 weeks old, 15–25 g), male Wistar rats (7–8 weeks old, 200–280 g), and female Brown Norway rats (7–8 weeks old, 110–160 g for OVA-induced airway hyperreactivity, 4–5 weeks old, 70–100 g for OVA-induced pulmonary eosinophilia) were purchased from Charles River Laboratories Japan, Inc. (Yokohama, Japan). Animals were given food and water available ad libitum. All experiments were carried out in accordance with the regulations of the company Animal Ethics Committee.

Chemicals

Effect of YM-341619 on STAT6 promoter gene activity

The treatment of FW4 cells with IL-4 (1 ng/ml) increased STAT6 luciferase gene activity nine-fold relative to un-stimulated cells (Fig. 2). YM-341619 (0.1-100 nM) inhibited the STAT6 luciferase gene activity in a concentration dependent manner, with an IC50 value of 1.5 nM.

Effect of YM-341619 on IL-4 and IFN-γ production, and GATA-3 and T-bet mRNA expression in T cells cultured with IL-4 or IL-12

T cells cultured with IL-4 in addition to anti-CD3 and CD28 antibody produced IL-4 by the final anti-CD3 antibody stimulation (Fig. 3A). However, T cells cultured with IL-12 in addition to anti-CD3 antibody did not produce

Discussion

In this study, we demonstrated that the novel compound, YM-341619, selectively suppressed the differentiation of naive T cells into the Th2 subset both in vitro and in vivo. YM-341619 reduced IL-4 production and GATA-3 expression on T cells cultured with IL-4 concentration-dependently within the range of 0.1 nM to 10 nM, but did not affect IFN-γ production or T-bet expression on T cells cultured with IL-12 (Fig. 4). IL-4 is a principle cytokine that drives naive CD4+ T cells to differentiate

Acknowledgements

We would like to thank Dr. Tadatsugu Taniguchi (Department of Immunology, University of Tokyo, Japan) for kindly providing the FW4 cells.

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