Requirement for Fucosyltransferase 2 in Allergic Airway Hyperreactivity and Mucus Obstruction

Abstract

Mucus hypersecretion is an important pathological problem in respiratory diseases. Mucus accumulates in the airways of people with asthma and contributes to airflow limitation by forming plugs that occlude airways. Current treatments have minimal effects on mucus or its chief components, the polymeric mucin glycoproteins MUC5AC and MUC5B. This treatment gap reflects a poor molecular understanding of mucins that could be used to determine how they contribute to airway obstruction. Because of the prominence of glycosylation as a defining characteristic of mucins, we investigated characteristics of mucin glycans in asthma and in a mouse model of allergic asthma. Mucin fucosylation was observed in asthma, and in healthy mice it was induced as part of a mucous metaplastic response to allergic inflammation. In allergically inflamed mouse airways, mucin fucosylation was dependent on the enzyme fucosyltransferase 2. Fut2 gene-deficient mice were protected from asthma-like airway hyperreactivity and mucus plugging. These findings provide mechanistic and translational links between observations in human asthma and a mouse model that may help improve therapeutic targeting of airway mucus.

Keywords: airways; asthma; glycosylation; mucin; mucus.

Figure 1.

Fucosyltransferase (FUT2)–mediated mucin fucosylation. Mucin fucosylation occurs via FUT2-mediated attachment of fucose (Fuc; red triangles) to galactose (Gal; yellow circles) glycans that extend from underlying Ser or Thr residues possessing O-linked GalNAc (yellow squares) with or without GlcNAc (blue squares) and Gal extensions. Fuc is transferred from a GDP-linked carrier to Gal via bond formation between carbon 1 of Fuc and carbon 2 of Gal, resulting in a Fuc(α1-2)Gal structure. GDP = guanosine diphosphate; GalNAc = N-acetylgalactosamine; GlcNAc = N-acetylglucosamine; Ser = serine; Thr = threonine.

Figure 2.

Fucosylation of airway polymeric mucins in human asthma and in a mouse model. (A and B) In a human airway, α2-Fuc was detected using the lectin Ulex europaeus agglutinin-1 (UEA1; yellow, 8 μg/ml), which labeled airway mucous cells and a mucus plug in a person who died of fatal asthma. MUC5AC (magenta, 1:1,000) and MUC5B (cyan, 1:500) were both detected and fucosylated. (C and D) In allergic mouse airways, Muc5b (cyan, 1:500), Muc5ac (magenta, 1:2,500), and α2-Fuc (UEA1, yellow, 8 μg/ml) labeling colocalize within airway epithelia. Monochrome (left) and pseudocolored individual label (right) versions are shown for human (C) and mouse (D) labels. Nuclei are stained with DAPI (blue). Scale bars: A, B, 100 μm; C, D, 10 μm. AOE = Aspergillus oryzae extract.

Figure 3.

Allergic mouse airway epithelial α2-fucosylation requires Fut2. (A and B) At baseline, α2-Fuc and Muc5ac are not detectable in airway epithelia in Fut2^+/+ (A) of Fut2^−/− (B) mice, but Muc5b is observed. (C and D) After AOE, Muc5b is sustained, while Muc5ac and α2-Fuc detection are both induced in Fut2^+/+ animals (C). In AOE-challenged Fut2^−/− mice, Muc5ac and Muc5b are sustained, but α2-Fuc detection is lost. Tissues were labeled with anti-Muc5b (cyan, 1:500), anti-Muc5ac (magenta, 1:2,500), biotinylated UEA1 (yellow, 8 μg/ml), and DAPI (blue). Scale bars: 100 μm; (inset) 40 μm.

Figure 4.

Fut2 is required for allergic airway hyperreactivity. (A–D) Dose–response curves to nebulized methacholine (MCh; 0.1–10 mg/ml) were generated in AOE-challenged and saline vehicle–challenged Fut2^+/+ (black) and Fut2^−/− (magenta) mice. Changes in total lung resistance (R_L; A and B) and conducting airway resistance (R_AW; C and D) in response to MCh were measured. Values are mean ± SEM with individual points per animal depicted by semitransparent shapes. For each animal, dose–response curves were fitted by log-linear best fit regression analysis, and slopes of regression lines were compared using one-way ANOVA. *P < 0.05 using Dunnett’s post hoc test for multiple comparisons relative to AOE-challenged Fut2^+/+ mice (P values are shown in B and D). Comparisons were made between AOE-challenged Fut2^+/+ mice (solid gray, black solid lines, n = 7 biological replicates), AOE-challenged Fut2^−/− mice (solid magenta, solid lines, n = 6 biological replicates), saline-challenged Fut2^+/+ mice (open gray, black dashed lines, n = 7 biological replicates for R_L, n = 6 for R_AW), and saline-challenged Fut2^−/− mice (open magenta, dashed lines, n = 8 biological replicates). Squares identify males, and circles identify females.

Figure 5.

Fut2 deficiency does not reduce inflammation in AOE-challenged mice. Lung lavage fluid was obtained from saline and AOE-challenged Fut2^+/+ (gray–black) and Fut2^−/− (magenta) mice. (A–C) AOE challenge resulted in increases in total numbers of leukocytes (A), eosinophils (B), and macrophages (C) in both Fut2^+/+ and Fut2^−/− mice compared with saline-challenged control animals. Kruskal-Wallis ANOVA was used to compare saline-challenged Fut2^+/+ mice (open gray shapes, n = 10 biological replicates), saline-challenged Fut2^−/− mice (open magenta shapes, n = 9 biological replicates), AOE-challenged Fut2^+/+ mice (solid gray shapes, n = 9 biological replicates), and AOE-challenged Fut2^−/− mice (solid magenta shapes, n = 10 biological replicates). *P < 0.05 using Dunn’s post hoc test for multiple comparisons (P values are shown). Squares identify males, and circles identify females. No significant differences were observed using sex as a biological variable.

Figure 6.

Fut2 is required for Muc5ac and Muc5b α2-fucosylation. Combined immunoblotting and lectin blotting was performed on neat lavage. Equal volumes of lavage fluid (27 μl) were loaded per well, separated on 1% SDS agarose gels, and transferred to polyvinylidene fluoride membranes. Membranes were probed with biotinylated UEA1 (1:1,000, 2 μg/ml) and either rabbit–anti-Muc5ac (1:2,000) or rabbit–anti-Muc5b (1:5,000). For secondary detection, Alexa 680–conjugated streptavidin and Alexa 800–conjugated labeled goat–antirabbit probes (Licor; 1:15,000) were applied. Monochrome images were acquired and pseudocolored magenta (Muc5ac), cyan (Muc5b), or yellow (α2-Fuc). Image overlays demonstrate UEA1 and mucin signal colocalization (white in merged panels).

Figure 7.

Fut2 deficiency reduces airway mucus plugging. After lung mechanics studies, lungs were fixed with methacarn to preserve mucus in airspaces. (A and B) Alcian blue–periodic acid–Schiff–stained tissues of Fut2^+/+ mice show mucin aggregated on airway surfaces (A) that was less prominent in Fut2^−/− animals (B). Scale bars, 10 μm. (C–E) Calculated mucus volumes (C), fractional mucin occlusion (D), and heterogeneous plugging (E) were significantly decreased in bronchial airways of AOE-challenged Fut2^−/− mice. Data in C and D are mean ± SEM for Fut2^+/+ (gray shapes, black lines, n = 5 mice) and Fut2^−/− (magenta shapes and lines, n = 6 mice). P values are shown; *P < 0.05 by two-tailed Mann-Whitney U tests. Data in E are mean ± SEM displayed on cumulative frequency distributions. Fractional occlusion means per animal were evaluated between Fut2^+/+ and Fut2^−/− mice using multiple unpaired t tests with two-stage step-up methods for multiple comparisons at a false discovery rate cutoff (q value) set at 0.05. Asterisk demonstrates significance, and q values are shown in E. In C–E, semitransparent shapes identify results from individual mice, squares identify males, and circles identify females. No significant differences were observed using sex as a biological variable. aw = airway lumen; epi = epithelium.