IL-12-STAT4-IFN-gamma axis is a key downstream pathway in the development of IL-13-mediated asthma phenotypes in a Th2 type asthma model

Abstract

IL-4 and IL-13 are closely related cytokines that are produced by Th2 cells. However, IL-4 and IL-13 have different effects on the development of asthma phenotypes. Here, we evaluated downstream molecular mechanisms involved in the development of Th2 type asthma phenotypes. A murine model of Th2 asthma was used that involved intraperitoneal sensitization with an allergen (ovalbumin) plus alum and then challenge with ovalbumin alone. Asthma phenotypes, including airway-hyperresponsiveness (AHR), lung inflammation, and immunologic parameters were evaluated after allergen challenge in mice deficient in candidate genes. The present study showed that methacholine AHR and lung inflammation developed in allergen-challenged IL-4-deficient mice but not in allergen-challenged IL-13-deficient mice. In addition, the production of OVA-specific IgG2a and IFN-gamma-inducible protein (IP)-10 was also impaired in the absence of IL-13, but not of IL-4. Lung-targeted IFN-gamma over-expression in the airways enhanced methacholine AHR and non-eosinophilic inflammation; in addition, these asthma phenotypes were impaired in allergen-challenged IFN-gamma-deficient mice. Moreover, AHR, non-eosinophilic inflammation, and IFN-gamma expression were impaired in allergen-challenged IL-12Rbeta2- and STAT4-deficient mice; however, AHR and non-eosinophilic inflammation were not impaired in allergen-challenged IL-4Ralpha-deficient mice, and these phenomena were accompanied by the enhanced expression of IL-12 and IFN-gamma. The present data suggest that IL-13-mediated asthma phenotypes, such as AHR and non-eosinophilic inflammation, in the Th2 type asthma are dependent on the IL-12-STAT4-IFN-gamma axis, and that these asthma phenotypes are independent of IL-4Ralpha-mediated signaling.

Figure 1

IL-13, but not IL-4, is essential for the development of Th2 type asthma phenotypes. Evaluations (n = 5 each group) were performed after allergen challenge. ^*, P < 0.05 compared to mice sensitized with PBS; ^**, P < 0.05 versus the other groups. (A) Methacholine AHR 24 h after the last allergen challenge of IL-13^-/- (left panel), IL-4^-/- (right panel), and the corresponding WT control mice. (B) BAL cellularity 48 h after the last allergen challenge. (C) Representative lung histology 48 h after the last allergen challenge (H&E staining, original magnification ×200). a, WT-PBS; b, WT_OVA/alum; c, IL-4^-/-_PBS; d, IL-4^-/-_OVA/alum; e, IL-13^-/-_PBS; f, IL-13^-/-_OVA/alum. (D) The levels of serum OVA-specific IgE (left panel) and IgG2a (right panel) measured 48 h after the last allergen challenge. (E) The levels of TGF-β1 and IP-10 in the BAL fluids 48 h after the last allergen challenge.

Figure 2

Transgenic over-expression of IFN-γ in the airways enhanced methacholine AHR and non-eosinophilic lung inflammation. Evaluations (n = 8 each group) were performed before and 7 days after the administration of doxycycline-containing water (dox). ^*, P < 0.05 compared to littermate WT mice; ^**, P < 0.05 versus the other groups. Methacholine AHR (A) and BAL cellularity (B) before and 7 days after the administration of dox to IFN-γ TG mice and their WT control littermates. (C) Representative lung histology (H&E staining, original magnification ×200). a, WT_no dox; b, IFN-γ (+)_no dox; c, IFN-γ (+)_dox 7 d.

Figure 3

IFN-γ plays key roles in the development of AHR and non-eosinophilic lung inflammation in the murine model of Th2 type asthma. For all figures, evaluation (n = 5 each group) was performed after allergen challenge. ^*, P < 0.05 compared to mice sensitized with PBS; ^**, P < 0.05 versus the other groups. (A) Methacholine AHR in IFN-γ^-/- and WT (BALB/c) mice 24 h after the last allergen challenge. (B) BAL cellularity (left panel) and representative lung histology in IFN-γ^-/- and WT (BALB/c) mice 48 h after the last allergen challenge. (H&E staining, original magnification ×400). a, WT_PBS; b, IFN-γ^-/-_PBS; c, WT_OVA/alum; d, IFN-γ^-/-_OVA/alum. (C) BAL IP-10 (left panel) and TGF-β1 (right panel) levels in IFN-γ^-/- and WT control mice 48 h after the last allergen challenge. (D) BAL IL-12 (left panel) and IL-13 (right panel) levels in IFN-γ^-/- and WT control mice 6 h after three rounds of allergen challenge.

Figure 4

AHR and non-eosinophilic lung inflammation are not impaired by the absence of IL-4Rα-mediated signaling in the Th2 asthma model. Evaluations (n = 5 each group) were performed twice after allergen challenge. ^*, P < 0.05 compared to mice sensitized with PBS; ^**, P < 0.05 versus the other groups. (A) Levels of cytokines in the BAL fluids of the IL-4Rα^-/- and WT (BALB/c) mice 6 h after three rounds of allergen challenge. (B) Methacholine AHR 24 h after the final allergen challenge. (C) BAL cellularity (left panel) and representative lung histology (right panel) in IL-4Rα^-/- and WT mice 48 h after the final allergen challenge. (H&E staining, original magnification ×200). a, WT_PBS; b, IL-4Rα^-/-_PBS; c, WT_OVA/alum; d, IL-4Rα^-/-_OVA/alum. (D) Levels of TGF-β1 (upper panel) and IP-10 (lower panel) 48 h after the final allergen challenge.

Figure 5

AHR and non-eosinophilic lung inflammation are impaired by the absence of IL-12Rβ2-mediated signaling in the Th2 asthma model. Evaluations (n = 5 each group) were performed twice after allergen challenge. ^*, P < 0.05 compared to mice sensitized with PBS; ^**, P < 0.05 versus the other groups. (A) Methacholine AHR of IL-12Rβ2^-/- and WT (C57BL/6) mice 24 h after the final allergen challenge. (B) BAL cellularity (left panel) and representative lung histology 48 h after the final allergen challenge. (H&E staining, original magnification ×200). a, WT_PBS; b, IL-12Rβ2^-/-_PBS; c, WT_OVA/alum; d, IL-12Rβ2^-/-_OVA/alum. (C) The levels of IP-10 (left panel) and TGF-β1 (right panel) in the BAL fluids of IL-12Rβ2^-/- and WT mice 6 h after three rounds of allergen challenge. (D) IFN-γ-producing CD4+ and CD8+ T cells in IL-12Rβ2^-/- and WT mice 6 h after three rounds of allergen challenge. (E) Levels of IP-10 (left panel), IL-12 (middle panel), and TGF-β1 (right panel) in the IL-12Rβ2^-/- and WT mice 48 h after the final allergen challenge.

Figure 6

STAT4-mediated signaling plays a key role in the development of AHR and non-eosinophilic inflammation in the Th2 asthma model. Evaluations (n = 5 each group) were performed twice 48 h after the final allergen challenge. ^*, P < 0.05 compared to mice sensitized with PBS; ^**, P < 0.05 versus the other groups; ^***, P < 0.05 versus the WT_PBS group. Methacholine AHR (A) and BAL cellularity (B) in STAT4^-/- and WT (BALB/c) mice. (H&E staining, original magnification ×100). a, WT_PBS; b, STAT4^-/-_PBS; c, WT_OVA/alum; d, STAT4^-/-_OVA/alum. (C) Levels of IL-13 (upper left panel), TGF-β1 (upper right panel), and IL-12 (lower panel) in the STAT4^-/- and WT control mice. (D) IFN-γ-producing CD11b+ cells in the lungs of the STAT4^-/- and WT control mice.

Figure 7

Hypothetical scheme for the development of Th2 type asthma phenotypes. When allergens are inhaled, airway dendritic cells (DCs) present allergens to effector Th2 cells, which produce cytokines, such as IL-4 and IL-13 (A). These cytokines induce lung infiltration of inflammatory cells, including eosinophils (eosinophilic inflammation), via chemokine actions (B). Recruited macrophages (M2 phenotype) and eosinophils produce TGF-β, which induces lung fibrosis and inhibits airway hyperresponsiveness (C). IL-13 also induces the production of IL-12 from DCs, possibly via IL-13Rα2-mediated signaling, and effector Th1 cells are generated via the IL-12-IL-12Rβ2-STAT4-mediated pathway (D). IFN-γ produced by effector Th1 cells induces lung infiltration of inflammatory cells other than eosinophils (non-eosinophilic inflammation) via chemokine actions (E). Recruited macrophages (M1 phenotype) produce mediators that inhibit the action of TGF-β, which results in the enhancement of airway hyperresponsiveness (F).