Lung Epithelial CYP1 Activity Regulates Aryl Hydrocarbon Receptor Dependent Allergic Airway Inflammation

Abstract

The lung epithelial barrier serves as a guardian towards environmental insults and responds to allergen encounter with a cascade of immune reactions that can possibly lead to inflammation. Whether the environmental sensor aryl hydrocarbon receptor (AhR) together with its downstream targets cytochrome P450 (CYP1) family members contribute to the regulation of allergic airway inflammation remains unexplored. By employing knockout mice for AhR and for single CYP1 family members, we found that AhR^-/- and CYP1B1^-/- but not CYP1A1^-/- or CYP1A2^-/- animals display enhanced allergic airway inflammation compared to WT. Expression analysis, immunofluorescence staining of murine and human lung sections and bone marrow chimeras suggest an important role of CYP1B1 in non-hematopoietic lung epithelial cells to prevent exacerbation of allergic airway inflammation. Transcriptional analysis of murine and human lung epithelial cells indicates a functional link of AhR to barrier protection/inflammatory mediator signaling upon allergen challenge. In contrast, CYP1B1 deficiency leads to enhanced expression and activity of CYP1A1 in lung epithelial cells and to an increased availability of the AhR ligand kynurenic acid following allergen challenge. Thus, differential CYP1 family member expression and signaling via the AhR in epithelial cells represents an immunoregulatory layer protecting the lung from exacerbation of allergic airway inflammation.

Keywords: CYP1B1; airway epithelial cells; aryl hydrocarbon receptor; cytochrome P450 enzyme; eosinophilia; lung allergy.

Figure 1

AhR and CYP1B1 deficiency aggravates pollen-induced allergic airway inflammation. (A) Experimental setup. Mice of indicated genotypes were exposed for five weeks to RWE extract. Wildtype mice exposed to PBS served as negative controls (PBS). (B) Total IgE (left) and Amb a 1-specific IgG1 (right) at day 0 and endpoint. (C) Total and differential BAL cell counts, (D) Th2 cell frequency, (E) CCL11, IL-4, IL-13, IFN-γ expression in whole lung tissue and (F) histological scores (left) and representative lung sections (right). Arrows: inflammatory infiltrate; arrowheads: mucus hypersecretion; scale bar: 100µm. Mean ± SEM, n=6-21 mice/group from up to 3 independent experiments. Histological scores: mean ± SD, (n=5). ANOVA with Tukey’s multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 2

AhR and CYP1B1 deficiency aggravates HDM-induced allergic airway inflammation. (A) Experimental setup. AhR^-/- (left) and CYP1B1^-/- (right) were exposed to house dust mite (HDM) as indicated. (B, G) Total IgE (left) and Der f-specific IgG1 (right) at endpoint. (C, H) Total and differential BAL cell counts, (D, I) Th2 cell frequency, (E, J) CCL11, IL-4, IL-13, IL-17A mRNA levels in whole lung tissue, (F, K) histological scores (left) and representative lung sections (right) at endpoint in AhR^-/- and CYP1B1^-/-, respectively. Arrows: inflammatory infiltrate; arrowheads: mucus hypersecretion; scale bar: 100µm. Mean ± SEM, n=5-10 mice/group/experiment from 2 independent experiments. Histological scores: mean ± SD, (n=5). Student’s unpaired two-tailed t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 3

Non-hematopoietic lung cells express CYP1B1. (A) CYP1B1 expression in CD45⁻ relative to CD45⁺ lung cells from WT, AhR^-/- and CYP1B1^-/- mice. Mean ± SEM of 2-6 mice/group. (B) Immune fluorescence staining of PBS- and HDM-challenged WT lungs or (C) human lung sections. Red: CYP1B1, Green: CC10, Blue: DAPI. Scale bar: 50μm or 20μm (insets). (D) NHBEs were stimulated for 6h with HDM extract (40µg/mL) in the presence of the AhR inhibitor CH-223191 (1µM/mL), IL-4 (50 ng/mL) or a combination of both. Results display top 25 upregulated and top 10 downregulated DEGs identified in the IL-4- and CH-223191-treated group relative to HDM only (center). These DEGs are also shown in NHBEs treated solely with IL-4 (left) or CH-223191 (right), respectively. Mean fold change of 4 independent donors NHBE, normal human bronchial epithelial cell.

Figure 4

Non-hematopoietic CYP1B1 expression prevents exacerbation of HDM-induced allergic airway inflammation. (A) Schematic representation of the generation of WT and CYP1B1^-/- bone marrow chimeras, which were subsequently treated with HDM or PBS (setup Figure 2A ). (B) Total IgE (left) and Der f-specific IgG1 (right) at endpoint. (C) Total and differential BAL cell counts, (D) Th2 cell frequency, (E) IL-4, IL-13, IFN-γ, IL-22, IL-17A protein levels in whole lung tissue, (F) histological scores (left) and representative lung sections (right). Arrows: inflammatory infiltrate; arrowheads: mucus hypersecretion; scale bar: 100µm. Mean ± SEM, n=4-13 mice/group from 2 independent experiments. Histological scores: mean ± SD, (n=5). ANOVA with Tukey’s multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 5

AhR regulates gene expression in airway epithelial cells of sensitized mice. (A) Total DEGs in AhR^-/- and CYP1B1^-/- epithelial cells (EC) relatively to WT from untreated or allergic (HDM) mice. (B, C) RNAseq analysis of sort-purified EC (CD45⁻CD31⁻EpCAM⁺live⁺) of AhR^-/- and CYP1B1^-/- mice (B) Heatmap of all genes differently expressed in AhR^-/- or CYP1B1^-/- versus WT EC at steady state and their expression in HDM-exposed animals. *p < 0.05, **p < 0.01, ***p < 0.001. (C) KEGG pathway analysis of DEGs between AhR and WT EC of HDM-exposed animals. Only pathways with at least 5 DEGs are shown.

Figure 6

Functional consequences of CYP1B1 deficiency in airway epithelial cells. CYP1A1 expression in total lung tissue from mice either (A) untreated, (B) sensitized to RWE, or (C) sensitized to HDM. (D) CYP1A1 expression in CD45^- cells isolated from HDM-sensitized mice of indicated genotypes. (E, F) EROD assay in total lung homogenates of indicated genotypes after 24 hour stimulation with HDM (10μg/ml) (E) or FICZ (10nM) (F). (G) EROD assay and (H) CYP1A1 expression of murine MTEC of the indicated genotypes after 24 hour stimulation with HDM (10μg/ml) or FICZ (10nM). Mean ± SEM of 4-20/group/condition [n=2 for CYP1A1^-/- and CYP1A2^-/-, (A)]. (I) Targeted LC-MS/MS measurement of tryptophane, kynurenine and kynurenic acid in lung homogenates of the indicated genotype with or without AAI. Mean ± SD; n=5/group/treatment. MTEC, mouse tracheobronchial epithelial cells. Student’s unpaired two-tailed t-test with Welch’s correction. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.