Genome-wide association mapping of acute lung injury in neonatal inbred mice

Abstract

Reactive oxygen species (ROS) contribute to the pathogenesis of many acute and chronic pulmonary disorders, including bronchopulmonary dysplasia (BPD), a respiratory condition that affects preterm infants. However, the mechanisms of susceptibility to oxidant stress in neonatal lungs are not completely understood. We evaluated the role of genetic background in response to oxidant stress in the neonatal lung by exposing mice from 36 inbred strains to hyperoxia (95% O2) for 72 h after birth. Hyperoxia-induced lung injury was evaluated by using bronchoalveolar lavage fluid (BALF) analysis and pathology. Statistically significant interstrain variation was found for BALF inflammatory cells and protein (heritability estimates range: 33.6-55.7%). Genome-wide association mapping using injury phenotypes identified quantitative trait loci (QTLs) on chromosomes 1, 2, 4, 6, and 7. Comparative mapping of the chromosome 6 QTLs identified Chrm2 (cholinergic receptor, muscarinic 2, cardiac) as a candidate susceptibility gene, and mouse strains with a nonsynonymous coding single-nucleotide polymorphism (SNP) in Chrm2 that causes an amino acid substitution (P265L) had significantly reduced hyperoxia-induced inflammation compared to strains without the SNP. Further, hyperoxia-induced lung injury was significantly reduced in neonatal mice with targeted deletion of Chrm2, relative to wild-type controls. This study has important implications for understanding the mechanisms of oxidative lung injury in neonates.

Keywords: bronchopulmonary dysplasia; cardiac; cholinergic receptor; inflammation; muscarinic 2; quantitative trait locus.

Figure 1.

Histopathologic and BALF responses to hyperoxia exposure in 129S1/SvImJ, C3H/HeJ, and PWD/PhJ neonates. A) BALF protein concentration. B) PMNs. C) Representative H&E-stained histopathology specimens from formalin-fixed neonatal lungs. Arrows show differences in peribronchiolar edema, vascular leakage, cell infiltrate, and alveolar septal thickness. AV, alveoli; BR, bronchus or bronchiole; BV, blood vessel. *P < 0.05 vs. indicated strain; ^#P < 0.05 vs. air-exposed control; Student-Newman-Keuls test.

Figure 2.

Strain distribution patterns of lung injury phenotypes from BALF after 72 h exposure to hyperoxia. A) PMNs as a percentage of BALF total cells. B) Macrophages per milliliter BALF. C) Epithelial cells per milliliter BALF. D) Protein concentration in BALF). Bars represent means ± sem (n=5–28/strain).

Figure 3.

Genome-wide haplotype association map of BALF PMNs in 36 inbred strains of neonatal mice exposed to hyperoxia. A) Manhattan plot for hyperoxia-induced BALF PMNs (percentage of BALF total cells). Cumulative genomic position is on the x axis; −log₁₀ P values are on the y axis. Circled rectangle (chromosome 6) identifies a locus with a −log₁₀ P value of 4.60, indicative of association with hyperoxia-induced PMN infiltration. B) Enlarged view of the chromosome 6 locus identified by GWA of the PMN response to hyperoxia in inbred strains of mice and the underlying gene Chrm2.

Figure 4.

Haplotype structure of Chrm2 and the association with susceptibility to hyperoxia-induced neutrophilia in BALF in inbred neonatal mice. A) Truncated haplotype structure of Chrm2 with synonymous (Cs) and nonsynonymous (Cn) coding SNPs in strains of mice with similar haplotype. Uppercase symbols represent validated nucleotides. Lowercase symbols represent imputed nucleotides. B) Mean number of PMNs in strains of mice with the Chrm2 haplotypes 1 (CCT; n=3 strains), 2 (GCC; n=17 strains), and 3 (CTT; n=14 strains). *P < 0.05, Student-Newman-Keuls test. C) Mean number of BALF PMNs for strains (n=21) homozygous for the rs30378838 C allele and strains (n=15) homozygous for the T allele. *P < 0.05; t test.

Figure 5.

Differential pulmonary responses to hyperoxia exposure in Chrm2^+/+ and Chrm2^−/− neonates. A) BALF protein concentrations. B) Representative H&E-stained histopathology images. Arrows indicate differences in alveolar edema and thickening of the bronchial epithelium and alveolar septae. AV, alveoli; BR, bronchus or bronchiole; BV, blood vessel. Bars represent means ± sem (n=6–9/group). *P < 0.05 vs. Chrm2^+/+; ^#P < 0.05 vs. air; 2-way ANOVA.