Effects of a Janus kinase inhibitor, pyridone 6, on airway responses in a murine model of asthma

doi:10.1016/j.bbrc.2010.11.104

Biochemical and Biophysical Research Communications

Volume 404, Issue 1, 7 January 2011, Pages 261-267

https://doi.org/10.1016/j.bbrc.2010.11.104 Get rights and content

Abstract

Th2 cytokines and their downstream Janus kinase (JAK)-signal transducer and activation of transcription (STAT) pathways play a critical role in allergic asthma. We studied the effects of a pan-JAK inhibitor, pyridone 6 (P6), on asthmatic responses in a mouse model and investigated the mechanism for its biological effects. Mice were sensitized and challenged by ovalbumin (OVA). P6 treatment during the challenge phase suppressed eosinophilia in bronchoalveolar lavage (BAL) fluids but did not affect airway hyperresponsiveness (AHR). To improve the efficacy of the JAK inhibitor, P6 was encapsulated in polylactic-coglycolic acid nanoparticles (P6-PLGA). P6-PLGA treatment just before OVA challenge suppressed both airway eosinophilia and AHR. Although the IL-13 levels in BAL fluids and the OVA-specific IgE levels in serum after the challenge phase treatment with P6-PLGA were similar to those after a sham treatment, the eotaxin levels in BAL fluids and lung mCLCA3/Gob-5 expression were decreased in P6-PLGA-treated mice. Interestingly, the local IL-13 levels and serum OVA-specific IgE were decreased, while IL-17-producing T cells were increased by P6-PLGA treatment during the sensitization plus challenge phases. In vitro, P6 strongly suppressed the differentiation of Th2 from naive CD4 T cells, but it partly enhanced Th17 differentiation. P6 potently suppressed IL-13-mediated STAT6 activation and mCLCA3/Gob-5 expression in mouse tracheal epithelial cells. These findings suggest that the JAK inhibitor P6 suppresses asthmatic responses by inhibiting Th2 inflammation and that application of PLGA nanoparticles improves the therapeutic potency of P6.

Research highlights

► Th2 cytokines and their downstream JAK–STAT pathways play a critical role in asthma. ► A JAK inhibitor pyridone 6 was encapsulated in nanoparticles. ► We studied the effects of pyridone 6 on asthmatic responses in a mouse model. ► Pyridone 6 treatment during allergen challenge suppressed asthmatic responses.

Introduction

Th2 cytokines, including IL-4, IL-5, IL-9, and IL-13, are essential for generating the pathophysiological features of asthma [1]. These cytokines bind to receptors at the cell surface and activate signal transduction pathways, including the Janus-kinase (JAK)-signal transducer and activation of transcription (STAT) pathway. Among Th2 cytokines, IL-13 is now considered particularly critical. IL-13 activates both the IL-4Rα and IL-13Rα1 chains to stimulate JAK1, JAK2, and Tyk2, leading to phosphorylation of STAT6. JAK–STAT pathways play a crucial role in Th2 responses and may be considered as novel targets for asthma [2].

In addition to Th2 cytokines, other cytokines, including IL-2, IL-6, IL-12, and IFN-γ, also activate the JAK–STAT pathways. These respective cytokines signal via JAK1/JAK3-STAT5, JAK1/JAK2/Tyk2-STAT3, JAK2/Tyk2-STAT4, and JAK1/JAK2-STAT1 [3]. A distinct set of cytokines promotes the differentiation processes for Th cell lineage: IL-12/IFN-γ for Th1; IL-4 for Th2; TGF-β/IL-6 (IL-21) for Th17; and TGF-β/IL-2 for regulatory T cells (Tregs) [4], [5]. IL-17, which is required during initial allergic sensitization, reduced Th2-dependent inflammation in established diseases in an animal model [6], whereas transfer of Th17 cells together with Th2 cells in naïve mice augmented airway inflammation [7]. Tregs suppress established airway inflammation and airway hyperresponsiveness in mouse models of allergic asthma [8]. Therefore, it is possible that the inhibition of JAK–STAT pathway might suppress the potentially beneficial immune response, and the effects of JAK–STAT inhibition on allergic asthma are not completely clear.

Here, we investigated the effects of pan-JAK inhibitor pyridone 6 (P6) on ovalbumin (OVA)-induced mouse models of allergic asthma. P6 competitively binds to the ATP-binding site of JAK1, JAK2, JAK3, and Tyk2 with strong inhibitory characteristics, as IC₅₀ for JAK1, JAK2, JAK3, and Tyk2 is 15 nM, 1 nM, 5 nM, and 1 nM, respectively, in vitro [9]. It has been reported that P6 is rapidly taken up by cells [10]. To improve the sustainability of the JAK inhibitor, P6 was encapsulated in biodegradable polylactic-coglycolic acid (PLGA) nanoparticles and administered to mice. The PLGA nanoparticle is a drug delivery system for sustained delivery of agents via protection of the drug encapsulated against enzymatic degradation [11].

Section snippets

Preparation of P6 and P6-encapsulated PLGA nanoparticles

P6 was either obtained from Sigma–Aldrich or synthesized according to the procedure described previously [9] by Fuji Molecular Planning Co. (Yokohama, Japan) with similar results. P6 was prepared as a stock solution of 100 mM DMSO. P6-encapsulated PLGA nanoparticles (P6-PLGA) were generated with an emulsion–solvent evaporation technique, as previously described [12], at Hosokawa Powder Technology Research Institute, Osaka, Japan. PLGA was composed of a polylactic acid to glycolic acid ratio of

Effect of treatment with unencapsulated P6 on allergic asthmatic responses

Th2 cytokines activate signal transduction pathways, including JAK–STAT signaling, and we first studied the effects of unencapsulated P6 on airway responses during the challenge phase of allergic asthma. Treatment with P6 during the OVA challenge phase significantly reduced eosinophilia in BAL fluids compared to the treatment with vehicle but failed to affect AHR (Fig. 1A and B). The concentration of eotaxin in BAL fluids was reduced, but neither IL-13 and IFN-γ nor the serum OVA-specific IgE

Discussion

In this study, we demonstrated that, during the challenge phase of allergic asthma, unencapsulated P6 treatment suppressed airway eosinophilia, and P6-PLGA treatment suppressed eosinophilia, AHR, and goblet cell hyperplasia. These findings suggest that P6-PLGA treatment during allergen challenge suppresses asthmatic phenotypes.

Application of PLGA nanoparticles improved the therapeutic potency of P6 in this asthma model. Unencapsulated P6 treatment inhibited airway eosinophilia but not AHR or

Acknowledgments

The authors thank Ms. Ayako Hashizume and Ms. Makiko Umemoto, M.Sc., for their technical assistance. This work was partially supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan and by the National Institute of Biomedical Innovation, Japan.

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The pan-JAK inhibitor P6 generally blocked cytokine-induced proliferation of Ba/F3/gp130 cells and variants thereof (Fig. 1B). We have compared 10 and 100 μm P6, because both concentrations were used previously (23, 24). Here we decided to use the lower concentration because it was enough to inhibit proliferation of Ba/F3-gp130 cells (Fig. 1B), albeit the higher concentration resulted in a more profound inhibition of all analyzed Janus kinases (Fig. S1C).
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Previous studies have indicated that nitrogen dioxide (NO₂) exposure could increase airway sensitivity to allergens for children. Recently, fetal stress was proposed as a crucial factor for allergic airway response occurring in offspring. Considering that there is inadequate evidence linking maternal NO₂ exposure to offspring airway sensitivity to allergens, pregnant Balb/c mice were exposed daily to 2.5 ppm NO₂ throughout the gestation period; then, the offspring were challenged to an allergen (ovalbumin, OVA) to evaluate airway sensitivity. For air + saline group and air + OVA group, offspring mice were maternally exposed to clean air followed by treatment with saline and OVA, respectively, in adulthood. For NO₂ + saline group and NO₂ + OVA group, offspring mice were maternally exposed to NO₂ followed by treatment with saline and OVA, respectively, in adulthood. The results showed that maternal NO₂ exposure increased the level of OVA-immunoglobulin (Ig) E in serum and caused airway hyper-responsiveness and pathological changes in offspring. Furthermore, maternal NO₂ exposure altered the expression of pro-inflammatory factors and impaired the T helper (Th) 1/Th2 balance. In addition, janus kinase)-signal transducer and activator of transcription 6 pathway participated in OVA-induced airway sensitivity of offspring. Our study showed that the potential risk of airway sensitivity to allergens in offspring is enhanced by maternal NO₂ exposure and proposed a possible mechanism for preventing, alleviating, and evaluating the outcomes in polluted environments.

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Effects of a Janus kinase inhibitor, pyridone 6, on airway responses in a murine model of asthma

Abstract

Research highlights

Introduction

Section snippets

Preparation of P6 and P6-encapsulated PLGA nanoparticles

Effect of treatment with unencapsulated P6 on allergic asthmatic responses

Discussion

Acknowledgments

Eur. J. Pharmacol.

Bioorg. Med. Chem. Lett.

Adv. Drug Deliv. Rev.

Int. J. Pharm.

J. Biol. Chem.

J. Allergy Clin. Immunol.

Immunity

Interleukin-13 in asthma pathogenesis

Immunol. Rev.

Series introduction. JAK–STAT signaling in human disease

J. Clin. Invest.

Induction and effector functions of T(H)17 cells

Nature

How diverse–CD4 effector T cells and their functions

J. Mol. Cell Biol.

Interleukin-17 is a negative regulator of established allergic asthma

J. Exp. Med.