Distinct roles of FOXA2 and FOXA3 in allergic airway disease and asthma

Abstract

Rationale: Increased production of mucus is a prominent feature of asthma. IL-13-driven mucous cell metaplasia is associated with decreased expression of the transcription factor FOXA2 and increased expression of the related transcription factor FOXA3 in animal and cell culture models.

Objectives: Establish how changes in FOXA2 and FOXA3 expression contribute to mucous metaplasia and determine whether FOXA2 and FOXA3 expression is altered in asthma.

Methods: Mice expressing a Foxa2 transgene in airway epithelial cells and mice deficient in Foxa3 were analyzed after allergen sensitization and challenge. Expression of FOXA2, FOXA3, MUC5AC, and the highly IL-13-inducible gene CLCA1 was analyzed in airway biopsies from subjects with asthma and control subjects.

Measurements and main results: Expression of a Foxa2 transgene reduced allergen-induced mucous metaplasia by 45% compared with control transgenic mice (P < 0.05) whereas inactivation of Foxa3 had no detectable effects on mucous metaplasia. Expression of FOXA2 was reduced in subjects with asthma and was negatively correlated with MUC5AC and CLCA1 levels in subjects with asthma. In contrast, FOXA3 expression was not significantly correlated with MUC5AC and was positively correlated with CLCA1.

Conclusions: Increasing Foxa2 expression reduced mucous metaplasia in an allergic mouse model. Subjects with asthma had decreased FOXA2 expression, suggesting that therapeutic approaches that increase FOXA2 expression or function could be beneficial for reducing mucus production in asthma. Unlike FOXA2, FOXA3 did not regulate mucous metaplasia.

Figure 1.

Expression of a Foxa2 transgene in airway epithelial cells. (A and B) FOXA2 protein (brown) was identified by immunohistochemistry in the lungs of (A) saline-challenged and (B) ovalbumin (OVA)-challenged nontransgenic mice and (C) Foxa2 transgenic (Tg) mice. Insets show regions of the epithelium at ×2 higher magnification. (D) Expression of Foxa2 transgene mRNA in lungs from saline- and ovalbumin-challenged nontransgenic control and Foxa2 Tg mice was measured by quantitative reverse transcription-polymerase chain reaction (RT-PCR) using PCR primers that amplify Foxa2 transgene mRNAs but not mRNAs derived from the endogenous murine Foxa2 gene. Results represent means ± SEM for two mice per group. *P < 0.05 compared with control mice. n.d. = Not detected.

Figure 2.

Increased lung FOXA3 expression in allergen-challenged mice. (A) Expression of Foxa3 mRNA in lungs from saline- and ovalbumin (OVA)-challenged BALB/c mice (open columns), C57BL/6 mice (solid columns), and FVB/N mice (shaded columns) was measured by quantitative reverse transcription-polymerase chain reaction (RT-PCR). Results represent means ± SEM for five mice per group. *P < 0.05 compared with saline-challenged mice. (B) Immunoblotting for FOXA3 protein in lung extracts from two saline- or OVA-challenged wild-type (Foxa3^+/+) and one Foxa3^−/− mice. n.s. = Nonspecific.

Figure 3.

Mucous metaplasia and MUC5AC transcription are decreased by FOXA2 but not by FOXA3. (A) Epithelial mucin stores, represented as means ± SEM of the volume of mucin referenced to the volume of the airway epithelium (Vv mu, epi), in allergen-challenged Foxa2 transgenic (Foxa2 Tg) mice (n = 9), nontransgenic (non-Tg) control mice (n = 9), and enhanced green fluorescent protein (EGFP) transgenic (EGFP-Tg) mice (n = 4). *P < 0.05 compared with non-Tg control mice; ^†P < 0.05 compared with EGFP-Tg mice. (B) Distribution of the volume of mucin-containing regions. The volume of periodic acid Schiff-stained regions within the epithelium was measured by the point-sampled intercept method. Foxa2 Tg mice (n = 4; circles) were compared with non-Tg wild-type control mice (n = 4; triangles). On average, 125 and 134 PAS-stained regions were measured in each nontransgenic and Foxa2 transgenic mouse, respectively. wt = wild type. (C) Epithelial mucin stores in allergen-challenged Foxa3^−/− mice and wild-type control mice (16 per group). (D) NCI-H292 cells were transfected with a MUC5AC promoter-luciferase reporter in combination with either a control (empty) expression vector (pcDNA3.1) or human FOXA2 or FOXA3 expression vector. MUC5AC promoter activity was determined as relative luciferase activity normalized to β-galactosidase activity. Values represent means ± SD from three independent experiments. *P < 0.05 compared with control cells transfected with empty vector.

Figure 4.

Other features of allergic airway disease are not affected in Foxa2 transgenic mice. (A) Serum ovalbumin (OVA)-specific IgE was measured in saline- and OVA-challenged nontransgenic control and Foxa2 transgenic (Foxa2 Tg) mice. (B) Airway inflammation was analyzed by counting macrophages (Mac), eosinophils (Eos), lymphocytes (Lymph), and neutrophils (Neut) in bronchoalveolar lavage fluid. (C) Airway reactivity to intravenously administered acetylcholine was measured with the Flexivent system. Results represent means ± SEM for six to eight mice per group. *P < 0.05, **P < 0.01 compared with saline-challenged mice; ^†P < 0.05 compared with OVA-challenged mice.

Figure 5.

Reduced airway epithelial FOXA2 staining in subjects with asthma is associated with increased mucin stores. Airway epithelial biopsies from five healthy control subjects and five subjects with mild or moderate asthma were stained with FOXA2 antiserum. (A) Ratios of FOXA2-stained (FOXA2+) to FOXA2-unstained (FOXA2–) nuclei were determined by analyzing all the epithelium within multiple biopsies from each subject (mean of 367 nuclei per subject). **P < 0.01 compared with healthy subjects. (B) Correlation between mucin stores and the ratios of FOXA2+ to FOXA2– nuclei for all subjects (five control subjects [triangles] and five subjects with asthma [circles]; R = 0.47, P = 0.027). Vv mu, epi = epithelial mucin stores, represented as means ± SEM of the volume of mucin referenced to the volume of the airway epithelium.

Figure 6.

Expression of FOXA2, FOXA3, MUC5AC, and CLCA1 mRNAs in bronchial epithelial cells from control subjects and subjects with asthma. Transcript levels in endobronchial brush samples (97 ± 3% epithelial cells) were measured with microarrays as previously reported (27). (A) FOXA2 mRNA expression in 28 control subjects (C) and 42 subjects with asthma (A) were compared by Wilcoxon rank-sum test. (B and C) Correlation between FOXA2 and MUC5AC (B: R = −0.39) or CLCA1 (C: R = −0.40) mRNA expression in samples from subjects with asthma. (D) FOXA3 mRNA expression in control subjects and subjects with asthma. NS = not significant. (E and F) Correlation between FOXA3 and MUC5AC (E: R = 0.30) or CLCA1 (F: R = 0.49) mRNA expression in samples from subjects with asthma.