Allergen-induced peribronchial fibrosis and mucus production mediated by IkappaB kinase beta-dependent genes in airway epithelium

Abstract

In response to inflammation or injury, airway epithelial cells express inducible genes that may contribute to allergen-induced airway remodeling. To determine the contribution of epithelial cell NF-kappaB activation to the remodeling response, we generated CC10-Cre(tg)/Ikkbeta(delta/delta) mice in which NF-kappaB signaling through IkappaB kinase beta (IKKbeta) is selectively ablated in the airway epithelium by conditional Cre-recombinase expression from the Clara cell (CC10) promoter. Repetitive ovalbumin challenge of mice deficient in airway epithelial IKKbeta prevented nuclear translocation of the RelA NF-kappaB subunit only in airway epithelial cells, resulting in significantly lower peribronchial fibrosis in CC10-Cre(tg)/Ikkbeta(delta/delta) mice compared with littermate controls as assessed by peribronchial trichrome staining and total lung collagen content. Levels of airway mucus, airway eosinophils, and peribronchial CD4+ cells in ovalbumin-challenged mice were also reduced significantly upon airway epithelial Ikkbeta ablation. The diminished inflammatory response was associated with reduced expression of NF-kappaB-regulated chemokines, including eotaxin-1 and thymus- and activation-regulated chemokine, which attract eosinophils and Th2 cells, respectively, into the airway. The number of peribronchial cells expressing TGF-beta1, as well as TGF-beta1 amounts in bronchoalveolar lavage, were also significantly reduced in mice deficient in airway epithelium IKKbeta. Overall, these studies show an important role for NF-kappaB regulated genes in airway epithelium in allergen-induced airway remodeling, including peribronchial fibrosis and mucus production.

Fig. 1.

Generation of mice lacking IKKβ in airway epithelium. Genotyping of CC10-Cre^tg/Ikkβ^Δ/Δ mice. CC10-Cre^tg mice (A) were crossed with Ikkβ^F/F mice (B) to generate, after doxycycline administration, CC10-Cre^tg/Ikkβ^Δ/Δ progeny in which the Ikkβ^F allele is selectively deleted in airway epithelium. (A1) In CC10-Cre^tg mice the CC10 promoter induces selective airway epithelial expression of rtTA. (A2) In the presence of doxycycline, rtTA binds to a tetO-containing promoter activating transcription of a transgene encoding Cre-recombinase in the airway epithelium. (B) Cre-recombinase cleaves at the loxP sites that flank exon 3 of the Ikkβ^F allele to produce the deleted Ikkβ^Δ allele, which is null for IKKβ expression. (C) RT-PCR analysis of CC10-Cre^tg/Ikkβ^F/F progeny (lane 3) indicates the presence of both transgenes contributed by the CC10-Cre^tg mice (lane 2), as well as the Ikkβ^F allele, contributed by the Ikkβ^F/F parents (lane 1). (D) TaqMan PCR studies showing that, in CC10-Cre^tg/Ikkβ^Δ/Δ mice, sequences specific to Ikkβ exon 3 were absent in airway epithelial cells. (E) Activation of NF-κB after OVA challenge: Immunofluorescence microscopy of bronchiole sections stained with anti-RelA Abs and nuclear counterstaining with SYTOX green. RelA is cytoplasmic (green) in airway epithelial cells (arrow) as well as in peribronchial mononuclear cells of non-OVA-challenged WT and CC10-Cre^tg/Ikkβ^Δ/Δ mice. OVA-challenged WT mice exhibit nuclear RelA staining (yellow) in both airway epithelial cells (arrow) and peribronchial mononuclear cells. In OVA-challenged CC10-Cre^tg/Ikkβ^Δ/Δ mice RelA remained cytoplasmic in most airway epithelial cells (arrow) but was nuclear in peribronchial cells, which were reduced in number in CC10-Cre^tg/Ikkβ^Δ/Δcompared with WT mice.

Fig. 2.

Peribronchial fibrosis in CC10-Cre^tg/Ikkβ^Δ/Δ mice. WT and CC10-Cre^tg/Ikkβ^Δ/Δ mice were kept unchallenged or were subjected to repetitive OVA challenge. Lungs were collected, and the extent of fibrosis was evaluated by trichrome staining (blue).

Fig. 3.

Quantitative analysis of airway fibrosis, TGF-β1, inflammation, mucus, and chemokines. WT and CC10-Cre^tg/Ikkβ^Δ/Δ mice were kept unchallenged or subjected to repetitive OVA challenge. (A) Lungs and BAL fluid were collected, and the extent of fibrosis was evaluated by lung trichrome staining and image analysis. (B) Lung mucus assessed by periodic acid/Schiff reagent staining. (C) BAL eosinophils revealed by Wright-Giemsa staining. (D) BAL eotaxin-1 measured by an ELISA. (E) BAL IL-5 measured by an ELISA. (F) Lung TGF-β1 measured by an ELISA. (G) BAL TARC measured by an ELISA. (H) Peribronchial CD4⁺ cells identified by immunostaining with an anti-CD4 Ab.