Functional genomic assessment of phosgene-induced acute lung injury in mice

Abstract

In this study, a genetically diverse panel of 43 mouse strains was exposed to phosgene and genome-wide association mapping performed using a high-density single nucleotide polymorphism (SNP) assembly. Transcriptomic analysis was also used to improve the genetic resolution in the identification of genetic determinants of phosgene-induced acute lung injury (ALI). We prioritized the identified genes based on whether the encoded protein was previously associated with lung injury or contained a nonsynonymous SNP within a functional domain. Candidates were selected that contained a promoter SNP that could alter a putative transcription factor binding site and had variable expression by transcriptomic analyses. The latter two criteria also required that ≥10% of mice carried the minor allele and that this allele could account for ≥10% of the phenotypic difference noted between the strains at the phenotypic extremes. This integrative, functional approach revealed 14 candidate genes that included Atp1a1, Alox5, Galnt11, Hrh1, Mbd4, Phactr2, Plxnd1, Ptprt, Reln, and Zfand4, which had significant SNP associations, and Itga9, Man1a2, Mapk14, and Vwf, which had suggestive SNP associations. Of the genes with significant SNP associations, Atp1a1, Alox5, Plxnd1, Ptprt, and Zfand4 could be associated with ALI in several ways. Using a competitive electrophoretic mobility shift analysis, Atp1a1 promoter (rs215053185) oligonucleotide containing the minor G allele formed a major distinct faster-migrating complex. In addition, a gene with a suggestive SNP association, Itga9, is linked to transforming growth factor β1 signaling, which previously has been associated with the susceptibility to ALI in mice.

Figure 1.

Mouse strains vary in sensitivity to phosgene-induced acute lung injury. Survival time was determined for 43 mouse strains. Female mice were exposed to 1.0 ppm phosgene for up to 24 hours, and survival times were recorded hourly. Values are means ± SE (n = 10 mice per strain; 6–8 wk old).

Figure 2.

Histological assessment of lung tissue from control SM/J mice (A), control 129X1/SvJ mice (B), phosgene-exposed SM/J mice (C and E), or phosgene-exposed 129X1/SvJ mice (D and F). Consistent with phosgene-induced acute lung injury, hemorrhagic pulmonary edema was more evident in the sensitive SM/J strain than in the resistant 129X1/SvJ strain. Female mice were exposed to filtered air (control) or to phosgene (1 ppm for 12 h) and anesthetized, and lung tissue was obtained. Tissues were fixed in formaldehyde, and 5-μm sections were prepared with hematoxylin and eosin stain. Bars indicate magnification.

Figure 3.

Genome-wide association mapping of mouse strains that vary in sensitivity to phosgene-induced acute lung injury. (A) Genome-wide association map for phosgene-induced acute lung injury in mice. The scatter (Manhattan) plot was generated by efficient mixed-models association corrected for population structure and displays the corresponding –log (P) association probability for the top 1,000 region-wide single nucleotide polymorphisms (SNPs) at the indicated chromosomal locations. (B) Exemplary genetic locus on chromosome 2 illustrating SNP density. Within the gene boundaries of protein tyrosine phosphatase, receptor T, four SNPs exceeded the significance threshold of −log P > 4.8, and 61 other SNPs exceeded the suggestive linkage threshold of −log P > 4.0.

Figure 4.

Assessment of the phenotypic difference in survival time between the strains produced by nonsynonymous SNP associations in functional domains of candidate genes. For each SNP, the mean survival time was determined for mice in strains carrying one or the other of the two alleles (e.g., mean survival time of 320 mice with C for rs30121304 was compared with that for 110 mice with T for rs30121304). The difference between these allelic groups was then compared with the difference of the mean survival time of the mouse strains at the phenotypic extremes (i.e., sensitive PERA/EiJ [n = 10 mice] and resistant NOD/ShiLtJ [n = 10 mice] mouse strains exposed to 1 ppm phosgene; total = 410–430 female mice). The predicted amino acid substitutions in the corresponding protein are contained in the following domains: ALOX5 (V645I: valine 645 isoleucine): lipoxygenase domain; GALNT11 (S544L: serine 544 leucine): ricin-type β-trefoil lectin domain; ITGA9 (L353M leucine 353 methionine) VCBS (Vibrio, Colwellia, Bradyrhizobium, and Shewanella) putative adhesion domain; MBD4 (D128N: aspartic acid 128 asparagine): methyl CpG-binding domain; and MBD4 (A466T: alanine 466 threonine) endonuclease III domain, respectively. The predicted consequences of amino acid substitutions would alter side chain polarity (rs37913166, rs31503102), side chain charge (rs30840549), or hydropathy index (rs30121304, rs37913166, rs46653588). The SNP identification “rs” number is indicated on the abscissa with sample size within each bar. Values are means ± SE. P values indicate significance of the difference between the allele means as determined by ANOVA with an all-pairwise multiple comparison procedure (Holm-Sidak method). Alox5 = arachidonate 5-lipoxygenase; Galnt11 = UDP-N-acetyl-α-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase 11; Itga9 = integrin α9; Mbd4 = methyl-CpG binding domain protein 4.

Figure 5.

Transcripts in enriched pathways that were altered in sensitive SM/J more than in resistant 129X1/J mice as determined by microarray analysis of lung mRNA after phosgene exposure. Microarray analysis was performed on mouse lung mRNA obtained at 0 (control), 6, and 12 hours during 1.0 ppm phosgene exposure (n = 5 arrays per strain per time), and significant differences were determined by Partek software (P < 0.05). Transcripts log 2 ≥ 1.0 (i.e., ≥ 2-fold) increased or log 2 ≤ −1.0 decreased in SM/J mouse lung but not significantly different in 129X1/SvJ mouse lung as compared with SM/J control (0 h) transcripts were analyzed using Database for Annotation, Visualization, and Integrated Discovery. The most enriched term in categories of Gene Ontogeny (GO) biological process, GO molecular function, or Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were selected. Ten transcripts with the greatest difference between strains in these pathways are displayed. (A) At 6 hours, the enriched pathways with increased transcripts in SM/J mouse lung (n = 751) included apoptosis, unfolded protein response, and cytokine–cytokine receptor binding. The enriched pathways with decreased transcripts in SM/J mouse lung (n = 731) included positive regulation of angiogenesis, transmembrane receptor activity, and ribosome. (B) At 12 hours, the enriched pathways with increased transcripts in SM/J mouse lung (n = 898) included apoptosis, GTPase regulator activity, and mitogen activated protein kinase (MAPK) signaling. The enriched pathways with decreased transcripts in SM/J mouse lung (n = 989) included microtubule-based process, cytoskeletal protein binding, and axon guidance.

Figure 5.

Transcripts in enriched pathways that were altered in sensitive SM/J more than in resistant 129X1/J mice as determined by microarray analysis of lung mRNA after phosgene exposure. Microarray analysis was performed on mouse lung mRNA obtained at 0 (control), 6, and 12 hours during 1.0 ppm phosgene exposure (n = 5 arrays per strain per time), and significant differences were determined by Partek software (P < 0.05). Transcripts log 2 ≥ 1.0 (i.e., ≥ 2-fold) increased or log 2 ≤ −1.0 decreased in SM/J mouse lung but not significantly different in 129X1/SvJ mouse lung as compared with SM/J control (0 h) transcripts were analyzed using Database for Annotation, Visualization, and Integrated Discovery. The most enriched term in categories of Gene Ontogeny (GO) biological process, GO molecular function, or Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were selected. Ten transcripts with the greatest difference between strains in these pathways are displayed. (A) At 6 hours, the enriched pathways with increased transcripts in SM/J mouse lung (n = 751) included apoptosis, unfolded protein response, and cytokine–cytokine receptor binding. The enriched pathways with decreased transcripts in SM/J mouse lung (n = 731) included positive regulation of angiogenesis, transmembrane receptor activity, and ribosome. (B) At 12 hours, the enriched pathways with increased transcripts in SM/J mouse lung (n = 898) included apoptosis, GTPase regulator activity, and mitogen activated protein kinase (MAPK) signaling. The enriched pathways with decreased transcripts in SM/J mouse lung (n = 989) included microtubule-based process, cytoskeletal protein binding, and axon guidance.

Figure 6.

Transcripts in the enriched nuclear factor (erythroid-derived 2)–like 2 (NFE2L2, also known as Nrf2)–mediated oxidative stress response pathway in the lungs of sensitive SM/J and resistant 129X1/SvJ mice during phosgene-induced acute lung injury. Increased transcripts at 6 hours in SM/J but not in 129X1/SvJ mice were determined by microarray analysis. Transcripts in this pathway were similar between strains at 12 hours and included heme oxygenase (decycling) 1 (HMOX1); glutathione S-transferase α1 (Ya) (GSTA1); activating transcription factor 3 (ATF3); glutamate-cysteine ligase, catalytic subunit (GCLC); NAD(P)H dehydrogenase, quinone 1 (NQO1); glutathione reductase (GSR); v-maf musculoaponeurotic fibrosarcoma oncogene family, protein F (avian) (MAFF); thioredoxin reductase 1 (TXNRD1); activating transcription factor 4 (ATF4); and glutamate-cysteine ligase, modifier subunit (GCLM). Values represent means ± SE (n = 6–8 female mice per strain per time) normalized to SM/J control mice (0 h).

Figure 7.

Transcripts in enriched pathways that were altered in resistant 129X1/SvJ more than in sensitive SM/J mice as determined by microarray analysis of lung mRNA after phosgene exposure. Microarray analysis was performed on mouse lung mRNA obtained at 0 (control), 6, and 12 hours during 1.0 ppm phosgene exposure (n = 5 arrays per strain per time), and significance (P < 0.05) was determined by Partek software. Transcripts log 2 ≥ 1.0 (i.e., ≥2-fold) increased or log 2 ≤ −1.0 decreased in 129X1/SvJ mouse lung but not significantly different in SM/J mouse lung as compared with SM/J control (0 h) transcripts were analyzed using Database for Annotation, Visualization, and Integrated Discovery (DAVID). The most significant enriched term in the categories of Gene Ontogeny (GO) biological process, GO molecular function, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were selected. Ten transcripts with the greatest difference between strains in these pathways are displayed. (A) At 6 hours, the enriched pathways with increased transcripts in 129X1/SvJ mouse lung (n = 1,016) included lymphocyte activation, calcium ion binding, and T-cell receptor signaling. The enriched pathways with decreased transcripts in 129X1/SvJ mouse lung (n = 947) included positive regulation of cell death, peptidase inhibitor, and complement and coagulation. (B) At 12 hours, the enriched pathways with increased transcripts in 129X1/SvJ mouse lung (n = 627) included defense mechanism, carbohydrate/pattern binding, and cell adhesion molecules (CAMs). The enriched pathways with decreased transcripts in 129X1/SvJ mouse lung (n = 954) included regulation of transcription, chemokine receptor, and DNA repair.

Figure 7.

Transcripts in enriched pathways that were altered in resistant 129X1/SvJ more than in sensitive SM/J mice as determined by microarray analysis of lung mRNA after phosgene exposure. Microarray analysis was performed on mouse lung mRNA obtained at 0 (control), 6, and 12 hours during 1.0 ppm phosgene exposure (n = 5 arrays per strain per time), and significance (P < 0.05) was determined by Partek software. Transcripts log 2 ≥ 1.0 (i.e., ≥2-fold) increased or log 2 ≤ −1.0 decreased in 129X1/SvJ mouse lung but not significantly different in SM/J mouse lung as compared with SM/J control (0 h) transcripts were analyzed using Database for Annotation, Visualization, and Integrated Discovery (DAVID). The most significant enriched term in the categories of Gene Ontogeny (GO) biological process, GO molecular function, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were selected. Ten transcripts with the greatest difference between strains in these pathways are displayed. (A) At 6 hours, the enriched pathways with increased transcripts in 129X1/SvJ mouse lung (n = 1,016) included lymphocyte activation, calcium ion binding, and T-cell receptor signaling. The enriched pathways with decreased transcripts in 129X1/SvJ mouse lung (n = 947) included positive regulation of cell death, peptidase inhibitor, and complement and coagulation. (B) At 12 hours, the enriched pathways with increased transcripts in 129X1/SvJ mouse lung (n = 627) included defense mechanism, carbohydrate/pattern binding, and cell adhesion molecules (CAMs). The enriched pathways with decreased transcripts in 129X1/SvJ mouse lung (n = 954) included regulation of transcription, chemokine receptor, and DNA repair.

Figure 8.

Transcript levels of candidate genes in 129X1/SvJ as compared with SM/J mouse lung. (A) Baseline transcript levels for 10 candidate genes with significant SNP associations (−log P ≥ 4.8). Transcript levels were determined by real-time qPCR (n = 8 mice per strain). Values are means ± SE (n = 8 mice per strain) normalized to ribosomal protein L32 (RPL32). *Significant difference (P < 0.05) between the resistant 129X1/SvJ and sensitive SM/J mouse strains at baseline as determined by ANOVA with an all-pairwise multiple comparison procedure (Holm-Sidak method). (B) Transcript levels of a candidate gene, zinc finger, AN1-type domain 4 (Zfand4), that was changed during phosgene exposure. Transcript levels were determined by real-time qPCR (n = 8 mice per strain). Values are means ± SE (n = 8 mice/strain) normalized to ribosomal protein L32 (RPL32). *Significantly different (P < 0.05) from sensitive SM/J mouse strain at baseline (0 h control), as determined by ANOVA with an all pairwise multiple comparison procedure (Holm-Sidak method). ^†Significant difference (P < 0.05) between the resistant 129X1/SvJ and sensitive SM/J mouse strains at the same time as determined by ANOVA with an all-pairwise multiple comparison procedure (Holm-Sidak method). (C) Transcript levels of zinc finger, AN1-type domain proteins in the lung of sensitive SM/J or resistant 129X1/SvJ mice after phosgene exposure. Transcript levels were determined by microarray analysis. Values are means ± SE (n = 8 mice per strain). Three transcripts (ZFAND2A, ZFAND4, and ZFAND5) increased more in the lung of the sensitive SM/J as compared with the resistant 129X1/SvJ, whereas other ZFAND transcripts did not differ between strains (ZFAND1) or were unchanged (ZFAND2B, ZFAND3, and ZFAND6) in either strain after phosgene exposure. *Significantly different (P < 0.05) from sensitive SM/J mouse strain at baseline (0 h control) as determined by ANOVA with an all-pairwise multiple comparison procedure (Holm-Sidak method). ^†Significant difference (P < 0.05) between the resistant 129X1/SvJ and sensitive SM/J mouse strains at the same time as determined by ANOVA with an all-pairwise multiple comparison procedure (Holm-Sidak method). ALOX5 = arachidonate 5-lipoxygenase; ATP1A1 ATPase = Na⁺/K⁺ transporting α1 polypeptide; HRH1 = histamine receptor H1; MARCH8 = membrane-associated ring finger (C3HC4) 8; PHACTR2 = phosphatase and actin regulator 2; PTPRT = protein tyrosine phosphatase, receptor T; RELN = Reelin; 5033411D12Rik = RIKEN cDNA 5033411D12 gene (homolog to CaiB/baiF CoA-transferase protein family, C7ORF10); PLXND1: plexin D1; ZFAND1–6 = zinc finger, AN1-type domains 1 through 6.

Figure 9.

SNP (rs215053185) in the ATPase, Na⁺/K⁺ transporting α1 polypeptide gene promoter region influences nuclear protein binding capacity. Electrophoretic mobility shift assay of nuclear protein extract prepared from mouse lung epithelial cells (MLE-15) and 25-mers (−50 to −74 bp 5′ from the start site) containing the A or G allele in the middle of a biotinylated oligonucleotide. The biotinylated 25-mer oligonucleotide with the G allele (lanes 7–12), in contrast to the A allele (lanes 1–6), formed a major distinct faster-migrating complex (middle band arrow 3), which is more effectively competed by unbiotinylated oligonucleotide containing the G variant (lanes 11 and 12) compared with the unbiotinylated oligonucleotide containing the A variant (lanes 9 and 10). The G allele is the minor allele (present in sensitive strains) and could result in the gain of a putative DNA binding site for transcription factor 12 (Tcf12), which is a transcriptional repressor. Mice with the rs215053185 A allele were resistant (mean survival time, 24.8 ± 1.0 h), whereas mice with the G allele were sensitive (mean survival time, 11.3 ± 0.5 h; P < 0.001).