Epithelial NF-κB orchestrates house dust mite-induced airway inflammation, hyperresponsiveness, and fibrotic remodeling

Abstract

NF-κB activation within the epithelium has been implicated in the pathogenesis of asthma, yet the exact role of epithelial NF-κB in allergen-induced inflammation and airway remodeling remains unclear. In the current study, we used an intranasal house dust mite (HDM) extract exposure regimen time course in BALB/c mice to evaluate inflammation, NF-κB activation, airway hyperresponsiveness (AHR), and airway remodeling. We used CC10-IκBαSR transgenic mice to evaluate the functional importance of epithelial NF-κB in response to HDM. After a single exposure of HDM, mRNA expression of proinflammatory mediators was significantly elevated in lung tissue of wild-type (WT) mice, in association with increases in nuclear RelA and RelB, components of the classical and alternative NF-κB pathway, respectively, in the bronchiolar epithelium. In contrast, CC10-IκBαSR mice displayed marked decreases in nuclear RelA and RelB and mRNA expression of proinflammatory mediators compared with WT mice. After 15 challenges with HDM, WT mice exhibited increases in inflammation, AHR, mucus metaplasia, and peribronchiolar fibrosis. CC10-IκBαSR transgenic mice displayed marked decreases in neutrophilic infiltration, tissue damping, and elastance parameters, in association will less peribronchiolar fibrosis and decreases in nuclear RelB in lung tissue. However, central airway resistance and mucus metaplasia remained elevated in CC10-IκBαSR transgenic mice, in association with the continued presence of lymphocytes, and partial decreases in eosinophils and IL-13. The current study demonstrates that following airway exposure with an asthma-relevant allergen, activation of classical and alternative NF-κB pathways occurs within the airway epithelium and may coordinately contribute to allergic inflammation, AHR, and fibrotic airway remodeling.

Figure 1. Analysis of cell totals and differentials in BAL of mice exposed to HDM

(A) Schematic depicting the time course of HDM exposure and euthanization. Instill: 50μg HDM, or PBS as the vehicle control were administered intranasally once at the days indicated via downward arrows. Mice were euthanized 2 h, 6 h or 24 h post a single challenge, 24 h after 3 challenges or 72 h after 5, 10, or 15 challenges, indicated by the upward arrows. (B) Mice (n=5/group/time point) were exposed to PBS or HDM according to the schematic in A, and BAL was assessed for neutrophils, eosinophils, lymphocytes, and macrophages. * p< 0.05 (ANOVA) compared to the PBS group at the same time point.

Figure 2. Assessment of nuclear localization of RelA and RelB in airway epithelium in response to HDM

BALB/c mice were exposed to PBS and HDM and harvested as indicated. De-paraffinized lung sections were incubated with antibodies directed against RelA or RelB, followed by fluorophore-conjugated secondary antibodies, and the nuclei were counterstained with Sytox Green. Images were captured by laser scanning confocal microscopy using identical instrument settings in all groups. Images are representative results of two independent experiments, n=5/group/time. RelA/RelB: red, DNA: green, Nuclear RelA/RelB: yellow. scale bar=50μm.

Figure 3. Nuclear RelA and RelB, and pro-inflammatory cytokine mRNA expression in CC10-NF-κB_SR mice following exposure to HDM

CC10-NF-κB_SR mice, or WT littermate controls were exposed to PBS or HDM for 2 h. (A) Nuclear localization of RelA and RelB was assessed in lung tissues as described in Fig. 2. Images are representative results of two independent experiments with 5 mice/group, RelA/RelB, red; DNA, green; Nuclear RelA/RelB, yellow, scale bar=50μm. (B) Assessment of mRNA expression for IL-33, GM-CSF, CCL20, KC, MIP-2, and IL-6 in homogenized lung tissue. Data were normalized to cyclophilin and are presented as relative expression. Values reflect 6 mice/group/time point. * p< 0.05 (ANOVA) compared to the PBS group at the same time point. † p<0.05 (ANOVA) compared to WT mice exposed to HDM

Figure 4. Assessment of HDM-induced inflamation, immunoglobins, T-cell cytokines, and nuclear RelB in WT or CC10-NF-κB_SR mice following 15 challenges with HDM

Assessment of (A) total cells and differential cell counts (B) and KC levels(C) in BAL. (D) Total IgG1 and IgE in serum. (E) IFNγ, IL-13, and IL-17A in homogenized tissue by ELISA. (F) Nuclear content of RelB in homogenized lung tissue. Histone H3 is shown as a loading control. Data represents 8 mice/group/time point. * p< 0.05 (ANOVA) compared to the PBS group at the same time point. † p<0.05 (ANOVA) compared to WT mice exposed to HDM.

Figure 5. Assessment of HDM-induced alterations in respiratory mechanics and airway remodeling in WT or CC10-NF-κB_SR mice following 15 challenges with HDM

(A) Evaluation of airway hyperresponsiveness via forced oscillation mechanics in response to 3.125, 12.5, and 25 mg methacholine. Shown are the parameters Newtonian resistance (Rn), Tissue damping (G) and Elastance (H) assessed via forced oscillation (B) Histopathological evaluation of tissue inflammation (H&E), mucus metaplasia (PAS) and peri-bronchiolar collagen deposition (M.T.). scale bar=50μm. (C) Analysis of Muc5ac mRNA expression in homogenized lung tissue. Data were normalized to cyclophilin and are presented as relative expression. (D) Quantification of mucus metaplasia. Bronchioles of similar size (n=3) were analyzed/mouse by two blinded scorers, and average scores presented as average units. (E) Assessment of collagen from the upper right lobe. (F) Quantification of peri-bronchiolar collagen deposition. Bronchioles of similar size (n=3) were analyzed/mouse by two blinded scorers, and average scores presented as average units. (G) Assessment of α-SMA immunohistochemistry, red=α-SMA, scale bar=1μm. (F) Quantification of α-SMA immunoreactivity. Bronchioles of similar size (n=3/mouse) were analyzed by two independent blinded scorers, and average scores presented as average units. Data represent 8 mice/group/time point. * p< 0.05 (ANOVA) compared to the PBS group at the same time point. † p<0.05 (ANOVA, Kruskal Wallis) compared to WT mice exposed to HDM.

Figure 6. Assessment of activation of classical and alternative NF-κB pathways in human lung epithelial cells exposed to HDM

(A) Primary human nasal epithelial (PHNE) cells or (B) Human bronchial epithelial (HBE) cells were exposed to PBS or 25 μg HDM once a day for consecutive days and harvested thereafter at either 72 h (PHNE) or 48 h (HBE). Cells were lysed for evaluation of NIK, p52, phospho RelA 536, by Western blot analyses. β-Actin (loading control). Right panels: Densitometric evaluation of Western blots shown in (A) PHNE (n=3 patients) or (B) HBE (n=3 experimental repeats). Results are expressed as arbitrary density and were normalized to corresponding β-Actin bands.