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Comparative Study
, 45 (4), 817-24

Strain-dependent Genomic Factors Affect Allergen-Induced Airway Hyperresponsiveness in Mice

Affiliations
Comparative Study

Strain-dependent Genomic Factors Affect Allergen-Induced Airway Hyperresponsiveness in Mice

Samir N P Kelada et al. Am J Respir Cell Mol Biol.

Abstract

Asthma is etiologically and clinically heterogeneous, making the genomic basis of asthma difficult to identify. We exploited the strain-dependence of a murine model of allergic airway disease to identify different genomic responses in the lung. BALB/cJ and C57BL/6J mice were sensitized with the immunodominant allergen from the Dermatophagoides pteronyssinus species of house dust mite (Der p 1), without exogenous adjuvant, and the mice then underwent a single challenge with Der p 1. Allergic inflammation, serum antibody titers, mucous metaplasia, and airway hyperresponsiveness were evaluated 72 hours after airway challenge. Whole-lung gene expression analyses were conducted to identify genomic responses to allergen challenge. Der p 1-challenged BALB/cJ mice produced all the key features of allergic airway disease. In comparison, C57BL/6J mice produced exaggerated Th2-biased responses and inflammation, but exhibited an unexpected decrease in airway hyperresponsiveness compared with control mice. Lung gene expression analysis revealed genes that were shared by both strains and a set of down-regulated genes unique to C57BL/6J mice, including several G-protein-coupled receptors involved in airway smooth muscle contraction, most notably the M2 muscarinic receptor, which we show is expressed in airway smooth muscle and was decreased at the protein level after challenge with Der p 1. Murine strain-dependent genomic responses in the lung offer insights into the different biological pathways that develop after allergen challenge. This study of two different murine strains demonstrates that inflammation and airway hyperresponsiveness can be decoupled, and suggests that the down-modulation of expression of G-protein-coupled receptors involved in regulating airway smooth muscle contraction may contribute to this dissociation.

Figures

Figure 1.
Figure 1.
Increased airway inflammation in C57BL/6J mice. (A) Differential cell counts in whole-lung lavage fluid. (B) Cytokines in whole-lung lavage fluid (n = 6–8/group/strain). Der p 1, house dust mite allergen Dermatophagoides pteronyssinus. MAC = macrophage, EOS = eosinophils, PMN = neutrophils, LYMPH = lymphocytes. ^Significantly different from control mice. *Significantly different between strains.
Figure 2.
Figure 2.
Increased Th2 responses in C57BL/6J mice after challenge with Der p 1. (A) Increased frequency of Th2 CD4+ lymphocytes in the lungs of Der p 1–challenged C57BL/6J mice. Left, Representative flow cytometry plots of CD4+ T cells. Numbers in plots represent the percentage of CD4+ T cells that were positive for each cytokine. Right, Summary data by strain (n = 4/group/strain). (B) Der p 1–specific cytokine release upon restimulation. Cells from mediastinal lymph nodes were isolated and cultured in the presence of PBS or 10 μg/ml Der p 1. Three days later, supernatants were collected and assayed. n = 6/group/strain. ^Significantly different from control mice. *Significantly different between strains.
Figure 3.
Figure 3.
Increased serum IgE and Der p 1–specific IgG1 in C57BL/6J mice. Left, Total IgE. Right, Der p 1–specific IgG1. Values for naive mice are shown for reference purposes (n = 6–8/group/strain). ^Significantly different from naive control mice. *Significantly different between strains.
Figure 4.
Figure 4.
Mucous metaplasia in both strains. (A) Hematoxylin and eosin (H&E), alcian blue–periodic acid–Schiff (AB-PAS), and periodic acid–fluorescent Schiff (PAFS)–stained sections of representative murine lungs. Scale bar, 150 μm. (B) Fluorescence measurements of mucus production, using PAFS staining (n = 6–8/group/strain). (C) Expression of mucin 5, subtypes A and C (Muc5ac) and chloride channel calcium activated 3 (Clca3, also known as Gob5) by quantitative RT-PCR. Data are presented as negative Δ Ct (−1 Delta Ct) to facilitate comparisons with data in A. Fold-change results are shown in Table E5 in the online supplement. ^Significantly different from control mice. *Significantly different between strains.
Figure 5.
Figure 5.
Resistance is increased in BALB/cJ mice, but decreased in C57BL/6J mice. (A) Total lung resistance (RL, cm H2O • second/ml) was measured using the forced oscillation technique (Flexivent). Gray vertical bars demarcate time periods (3 minutes each) spent measuring resistance at each concentration of methacholine. (B) Area under the curve (AUC) analysis of resistance data (n = 10–12/group/strain). ^Significantly different from control mice. *Significantly different between strains.
Figure 6.
Figure 6.
Shared and distinct patterns of lung gene expression in BALB/cJ and C57BL/6J mice. The numbers in the venn diagram indicate the number of genes that were differentially expressed by Der p 1 treatment, and the size of the circles is proportional to the number of genes in each group. For the full list of differentially expressed genes, see Table E2 (n = 10–12/group/strain).
Figure 7.
Figure 7.
The M2 muscarinic receptor (M2R) is expressed in airway smooth muscle and is decreased after challenge with Der p 1. (A) Immunofluorescent detection of M2R in airway smooth muscle. Lung sections from C57BL/6J mice were stained using antibodies for α–smooth muscle actin (α-SMA) and M2R. Note the colocalization of α-SMA and the M2R in airway, but not in vascular smooth muscle. Scale bar, 100 μm. Similar results were obtained with BALB/cJ mice. (B) Western blot analysis of M2R expression in saline-challenged and Der p 1–challenged C57BL/6J mice. Densitometric analysis (right) shows a 57% reduction in expression, attributable to challenge with Der p 1. ^Significantly different from control mice. DAPI, 4′,6-diamidino-2-phenylindole; Gapdh, glyceraldehyde-3-phosphate dehydrogenase.

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