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.
ARDS; countermeasures; genetics; sodium absorption; lipoxygenase
In the mouse lung, Escherichia coli LPS can decrease surfactant protein–B (SFTPB) mRNA and protein concentrations. LPS also regulates the expression, synthesis, and concentrations of a variety of gene and metabolic products that inhibit SFTPB gene expression. The purpose of the present study was to determine whether LPS acts directly or indirectly on pulmonary epithelial cells to trigger signaling pathways that inhibit SFTPB expression, and whether the transcription factor CCAAT/enhancer binding protein (C/EBP)–β (CEBPB) is a downstream inhibitory effector. To investigate the mechanism of SFTPB repression, the human pulmonary epithelial cell lines NCI-H441 (H441) and NCI-H820 (H820) and the mouse macrophage-like cell line RAW264.7 were treated with LPS. Whereas LPS did not decrease SFTPB transcripts in H441 or H820 cells, the conditioned medium of LPS-treated RAW264.7 cells decreased SFTPB transcripts in H441 and H820 cells, and inhibited SFTPB promoter activity in H441 cells. In the presence of neutralizing anti–tumor necrosis factor (TNF) antibodies, the conditioned medium of LPS-treated RAW264.7 cells did not inhibit SFTPB promoter activity. In H441 cells treated with recombinant TNF protein, SFTPB transcripts decreased, whereas CEBPB transcripts increased and the transient coexpression of CEBPB decreased SFTPB promoter activity. Further, CEBPB short, interfering RNA increased basal SFTPB transcripts and countered the decrease of SFTPB transcripts by TNF. Together, these findings suggest that macrophages participate in the repression of SFTPB expression by LPS, and that macrophage-released cytokines (including TNF) regulate the transcription factor CEBPB, which can function as a downstream transcriptional repressor of SFTPB gene expression in pulmonary epithelial cells.
acute respiratory distress syndrome; sepsis; acute lung injury; TNF; surfactant
Although strides have been made to reduce ventilator-induced lung injury (VILI), critically ill patients can vary in sensitivity to VILI, suggesting gene–environment interactions could contribute to individual susceptibility. This study sought to uncover candidate genes associated with VILI using a genome-wide approach followed by functional analysis of the leading candidate in mice. Alveolar–capillary permeability after high tidal volume (HTV) ventilation was measured in 23 mouse strains, and haplotype association mapping was performed. A locus was identified on chromosome 15 that contained ArfGAP with SH3 domain, ankyrin repeat and PH domain 1 (Asap1), adenylate cyclase 8 (Adcy8), WNT1-inducible signaling pathway protein 1 (Wisp1), and N-myc downstream regulated 1 (Ndrg1). Information from published studies guided initial assessment to Wisp1. After HTV, lung WISP1 protein increased in sensitive A/J mice, but was unchanged in resistant CBA/J mice. Anti-WISP1 antibody decreased HTV-induced alveolar–capillary permeability in sensitive A/J mice, and recombinant WISP1 protein increased HTV-induced alveolar–capillary permeability in resistant CBA/J mice. HTV-induced WISP1 coimmunoprecipitated with glycosylated Toll-like receptor (TLR) 4 in A/J lung homogenates. After HTV, WISP1 increased in strain-matched control lungs, but was unchanged in TLR4 gene–targeted lungs. In peritoneal macrophages from strain-matched mice, WISP1 augmented LPS-induced TNF release that was inhibited in macrophages from TLR4 or CD14 antigen gene–targeted mice, and was attenuated in macrophages from myeloid differentiation primary response gene 88 gene–targeted or TLR adaptor molecule 1 mutant mice. These findings support a role for WISP1 as an endogenous signal that acts through TLR4 signaling to increase alveolar–capillary permeability in VILI.
genome-wide association study; acute lung injury; acute respiratory distress syndrome
The genetic basis for the underlying individual susceptibility to chlorine-induced acute lung injury is unknown. To uncover the genetic basis and pathophysiological processes that could provide additional homeostatic capacities during lung injury, 40 inbred murine strains were exposed to chlorine, and haplotype association mapping was performed. The identified single-nucleotide polymorphism (SNP) associations were evaluated through transcriptomic and metabolomic profiling. Using ≥ 10% allelic frequency and ≥ 10% phenotype explained as threshold criteria, promoter SNPs that could eliminate putative transcriptional factor recognition sites in candidate genes were assessed by determining transcript levels through microarray and reverse real-time PCR during chlorine exposure. The mean survival time varied by approximately 5-fold among strains, and SNP associations were identified for 13 candidate genes on chromosomes 1, 4, 5, 9, and 15. Microarrays revealed several differentially enriched pathways, including protein transport (decreased more in the sensitive C57BLKS/J lung) and protein catabolic process (increased more in the resistant C57BL/10J lung). Lung metabolomic profiling revealed 95 of the 280 metabolites measured were altered by chlorine exposure, and included alanine, which decreased more in the C57BLKS/J than in the C57BL/10J strain, and glutamine, which increased more in the C57BL/10J than in the C57BLKS/J strain. Genetic associations from haplotype mapping were strengthened by an integrated assessment using transcriptomic and metabolomic profiling. The leading candidate genes associated with increased susceptibility to acute lung injury in mice included Klf4, Sema7a, Tns1, Aacs, and a gene that encodes an amino acid carrier, Slc38a4.
ARDS; countermeasures; glutamine; genetics; metabolomics
The murine surfactant-associated protein B (Sftpb) gene promoter, spanning nucleotides −653 to +42, is composed of functionally distinct proximal and distal regions. Although both regions contain consensus/putative activator protein 1 (AP-1) sites, the distal, but not the proximal, region mediates the inhibition by jun proto-oncogene (JUN) of Sftpb promoter activity. In transient cotransfection assays, JUN inhibited the luciferase reporter activity of plasmid constructs containing Sftpb promoter fragments that lacked the distal putative AP-1 site, indicating that another regulatory motif mediates JUN-dependent inhibition. Electrophoretic mobility shift assays and in silico analyses identified a DNA target sequence (Sftpb nucleotides −339 to −316) and transcription factors that regulate Sftpb promoter activity. The identified sequence contains a CCAAT/enhancer-binding protein (C/EBP) consensus recognition element. Mutation of the site reduced Sftpb promoter activity and sensitivity to inhibition by JUN. Purified recombinant JUN, which did not recognize the −339 to −316 target sequence when added alone, supershifted the mobility of in vitro translated C/EBP-α and C/EBP-β proteins complexed with the identified cis-regulatory element. These findings support the idea that heterodimerization between JUN and C/EBP-α and/or C/EBP-β targets JUN to the Sftpb promoter, thereby mediating its inhibitory regulatory role.
surfactant protein B; acute lung injury; gene regulation; pulmonary surfactant metabolism dysfunction type 1; pulmonary alveolar proteinosis
An integral membrane protein, Claudin 5 (CLDN5), is a critical component of endothelial tight junctions that control pericellular permeability. Breaching of endothelial barriers is a key event in the development of pulmonary edema during acute lung injury (ALI). A major irritant in smoke, acrolein can induce ALI possibly by altering CLDN5 expression. This study sought to determine the cell signaling mechanism controlling endothelial CLDN5 expression during ALI. To assess susceptibility, 12 mouse strains were exposed to acrolein (10 ppm, 24 h), and survival monitored. Histology, lavage protein, and CLDN5 transcripts were measured in the lung of the most sensitive and resistant strains. CLDN5 transcripts and phosphorylation status of forkhead box O1 (FOXO1) and catenin (cadherin-associated protein) beta 1 (CTNNB1) proteins were determined in control and acrolein-treated human endothelial cells. Mean survival time (MST) varied more than 2-fold among strains with the susceptible (BALB/cByJ) and resistant (129X1/SvJ) strains (MST, 17.3 ± 1.9 h vs. 41.4 ± 5.1 h, respectively). Histological analysis revealed earlier perivascular enlargement in the BALB/cByJ than in 129X1/SvJ mouse lung. Lung CLDN5 transcript and protein increased more in the resistant strain than in the susceptible strain. In human endothelial cells, 30 nM acrolein increased CLDN5 transcripts and increased p-FOXO1 protein levels. The phosphatidylinositol 3-kinase inhibitor LY294002 diminished the acrolein-induced increased CLDN5 transcript. Acrolein (300 nM) decreased CLDN5 transcripts, which were accompanied by increased FOXO1 and CTNNB1. The phosphorylation status of these transcription factors was consistent with the observed CLDN5 alteration. Preservation of endothelial CLDN5 may be a novel clinical approach for ALI therapy.
ARDS; perivascular edema; vascular permeability; smoke inhalation; carboxyl stress
The etiology of acute lung injury is complex and associated with numerous, chemically diverse precipitating factors. During acute lung injury in mice, one key event is epithelial cell injury that leads to reduced surfactant biosynthesis. We have previously reported that transgenic mice that express transforming growth factor α (TGFA) in the lung were protected during nickel-induced lung injury. Here, we find that the mechanism by which TGFA imparts protection includes maintenance of surfactant-associated protein B (SFTPB) transcript levels and epidermal growth factor receptor–dependent signaling in distal pulmonary epithelial cells. This protection is complex and not accompanied by a diminution in inflammatory mediator transcripts or additional stimulation of antioxidant transcripts. In mouse lung epithelial (MLE-15) cells, microarray analysis demonstrated that nickel increased transcripts of genes enriched in MTF1, E2F-1, and AP-2 transcription factor–binding sites and decreased transcripts of genes enriched in AP-1–binding sites. Nickel also increased Jun transcript and DNA-binding activity, but decreased SFTPB transcript. Expression of SFTPB under the control of a doxycycline-sensitive promoter increased survival during nickel-induced injury as compared with control mice. Together, these findings support the idea that maintenance of SFTPB expression is critical to survival during acute lung injury.
adult respiratory distress syndrome; innate immunity; chemokine; surfactant
We recently reported that induction of metallothionein (MT) was critical in limiting nickel (Ni)-induced lung injury in intact mice. Nonetheless, the mechanism by which Ni induces MT expression is unclear. We hypothesized that the ability of Ni to mobilize zinc (Zn) may contribute to such regulation and therefore, we examined the mechanism for Ni-induced MT2A expression in human airway epithelial (BEAS-2B) cells. Ni induced MT2A transcript levels and protein expression by 4 hours. Ni also increased the activity of a metal response element (MRE) promoter luciferase reporter construct, suggesting that Ni induces MRE binding of the metal transcription factor (MTF-1). Exposure to Ni resulted in the nuclear translocation of MTF-1, and Ni failed to induce MT in mouse embryonic fibroblasts lacking MTF-1. As Zn is the only metal known to directly bind MTF-1, we then showed that Ni increased a labile pool of intracellular Zn in cells as revealed by fluorescence-activated cell sorter using the Zn-sensitive fluorophore, FluoZin-3. Ni-induced increases in MT2A mRNA and MRE-luciferase activity were sensitive to the Zn chelator, TPEN, supporting an important role for Zn in mediating the effect of Ni. Although neither the source of labile Zn nor the mechanism by which Ni liberates labile Zn was apparent, it was noteworthy that Ni increased intracellular reactive oxygen species (ROS). Although both N-acetyl cysteine (NAC) and ascorbic acid (AA) decreased Ni-induced increases in ROS, only NAC prevented Ni-induced increases in MT2A mRNA, suggesting a special role for interactions of Ni, thiols, and Zn release.
nickel; metallothionein; zinc; epithelium
Chronic obstructive pulmonary disease (COPD), a global public health problem, is characterized by progressive difficulty in breathing, with increased mucin production, especially in the small airways. Acrolein, a constituent of cigarette smoke and an endogenous mediator of oxidative stress, increases airway mucin 5, subtypes A and C (MUC5AC) production; however, the mechanism remains unclear. In this study, increased mMUC5AC transcripts and protein were associated with increased lung matrix metalloproteinase 9 (mMMP9) transcripts, protein, and activity in acrolein-exposed mice. Increased mMUC5AC transcripts and mucin protein were diminished in gene-targeted Mmp9 mice [Mmp9(-/-)] or in mice treated with an epidermal growth factor receptor (EGFR) inhibitor, erlotinib. Acrolein also decreased mTissue inhibitor of metalloproteinase protein 3 (an MMP9 inhibitor) transcript levels. In a cell-free system, acrolein increased pro-hMMP9 cleavage and activity in concentrations (100–300 nM) found in sputum from subjects with COPD. Acrolein increased hMMP9 transcripts in human airway cells, which was inhibited by an MMP inhibitor, EGFR-neutralizing antibody, or a mitogen-activated protein kinase (MAPK) 3/2 inhibitor. Together these findings indicate that acrolein can initiate cleavage of pro-hMMP9 and EGFR/MAPK signaling that leads to additional MMP9 formation. Augmentation of hMMP9 activity, in turn, could contribute to persistent excessive mucin production.
mucus; COPD; matrix metalloproteinase; cigarette smoke; oxidative stress
Acute lung injury (ALI) is a devastating condition resulting from diverse causes. Genetic studies of human populations indicate that ALI is a complex disease with substantial phenotypic variance, incomplete penetrance, and gene–environment interactions. To identify genes controlling ALI mortality, we previously investigated mean survival time (MST) differences between sensitive A/J (A) and resistant C57BL/6J (B) mice in ozone using quantitative trait locus (QTL) analysis. MST was significantly linked to QTLs (Aliq1-3) on chromosomes 11, 13, and 17, respectively. Additional QTL analyses of separate and combined backcross and F2 populations supported linkage to Aliq1 and Aliq2, and established significance for previously suggestive QTLs on chromosomes 7 and 12 (named Aliq5 and Aliq6, respectively). Decreased MSTs of corresponding chromosome substitution strains (CSSs) verified the contribution of most QTL-containing chromosomes to ALI survival. Multilocus models demonstrated that three QTLs could explain the MST difference between progenitor strains, agreeing with calculated estimates for number of genes involved. Based on results of QTL genotype analysis, a double CSS (B.A-6,11) was generated that contained Aliq1 and Aliq4 chromosomes. Surprisingly, MST and pulmonary edema after exposure of B.A-6,11 mice were comparable to B mice, revealing an unpredicted loss of sensitivity compared with separate CSSs. Reciprocal congenic lines for Aliq1 captured the corresponding phenotype in both background strains and further refined the QTL interval. Together, these findings support most of the previously identified QTLs linked to ALI survival and established lines of mice to further resolve Aliq1.
acute respiratory distress syndrome; chromosome substitution strain; congenic; mean survival; pulmonary edema
Expression of transforming growth factor α (TGF-α) in the respiratory epithelium of transgenic mice caused pulmonary fibrosis, cachexia, pulmonary hypertension, and altered lung function. To identify genes and molecular pathways mediating lung remodeling, mRNA microarray analysis was performed at multiple times after TGF-α expression and revealed changes consistent with a role for TGF-α in the regulation of extracellular matrix and vasculogenesis. Transcripts for extracellular matrix proteins were augmented along with transcripts for genes previously identified to have roles in pulmonary fibrosis, including tenascin C, osteopontin, and serine (or cysteine) peptidase inhibitor, clade F, member 1. Transcripts regulating vascular processes including endothelin receptor type B, endothelial-specific receptor tyrosine kinase, and caveolin, caveolae protein 1 were decreased. When TGF-α expression was no longer induced, lung remodeling partially reversed and lung function and pulmonary hypertension normalized. Transcripts increased during resolution included midkine, matrix metalloproteinase 2, and hemolytic complement. Hierarchical clustering revealed that genes regulated by TGF-α were similar to those altered in the lungs of patients with idiopathic pulmonary fibrosis. These studies support a role for epithelial cell–derived TGF-α in the regulation of processes that alter the airway and vascular architecture and function.
epidermal growth factor receptor; idiopathic pulmonary fibrosis; vasculogenesis; angiogenesis; interstitial lung disease
Often fatal, acute lung injury has a complicated etiology. Previous studies from our laboratory in mice have demonstrated that survival during acute lung injury is a complex trait governed by multiple loci. We also found that the increase in metallothionein (MT) is one of the greatest noted in transcriptome-wide analyses of gene expression. To assess the role of MT in nickel-induced acute lung injury, the survival of Mt-transgenic, Mt1/2(+/+), and Mt1/2(+/+) mice was compared. Pulmonary inflammation and global gene expression were compared in Mt1/2(+/+) and Mt1/2(+/+) mice. Gene-targeted Mt1/2(+/+) mice were more susceptible than Mt1/2(+/+) mice to nickel-induced inflammation, surfactant-associated protein B transcript loss, and lethality. Similarly, Mt-transgenic mice exhibited increased survival. MAPPFinder analyses also noted significant decreases in genes involved in protein processing (e.g., ubiquitination, folding), which were greater in Mt1/2(+/+) mice as compared with Mt1/2(+/+) mice early in the progression of acute lung injury, possibly due to a zinc-mediated transcript destabilization. In contrast, transcript levels of genes associated with the inflammatory response, extracellular matrix regulation, and coagulation/fibrinolysis were increased more in Mt1/2(+/+) mice as compared with Mt1/2(+/+) mice late in the development of acute lung injury. Thus, MT ultimately improves survival in the progression of acute lung injury in mice. Transcriptome-wide analysis suggests that this survival may be mediated through changes in the destabilization of transcripts associated with protein processing, the subsequent augmentation of transcripts controlling inflammation, extracellular matrix regulation, coagulation/fibrinolysis, and disruption of surfactant homeostasis.
microarray; surfactant; inflammation; fibrinolysis; extracellular matrix
Previous studies have shown that mice deficient in the tyrosine kinase domain (TK−/−) of the receptor Mst1r have an increased susceptibility to nickel (Ni)-induced acute lung injury (ALI). Mst1r TK−/− mice have decreased survival times, alterations in cytokine and nitric oxide regulation, and an earlier onset of pulmonary pathology compared with control mice, suggesting that Mst1r signaling, in part, may regulate the response to ALI. To examine the role of Mst1r in ALI in more detail, we compared the gene expression profiles of murine lung mRNA from control and Mst1r TK−/− mice at baseline and after 24 h of particulate Ni sulfate exposure. Microarray analyses showed a total of 343 transcripts that were significantly changed, either by Ni treatment, or between genotypes. Genes responsible for inflammation, edema, and lymphocyte function were altered in the Mst1r TK−/− mice. Interestingly, the genes for several granzymes were increased in Mst1r TK−/− mice before Ni exposure, compared with controls. In addition, the Mst1r TK−/− lungs showed clusters of cells near the vascular endothelium and airways. Immunohistochemistry indicates these clusters are composed of macrophages, T cells, and neutrophils, and that the clusters display granzyme protein production. These results suggest that Mst1r signaling may be involved in the regulation of macrophage and T-lymphocyte activation in vivo during injury. This assessment of gene expression indicates the importance of genetic factors in contributing to lung injury, and points to strategies for intervention in the progression of inflammatory diseases.
acute lung injury; gene expression arrays; hepatocyte growth factor–like protein; Mst1r; Ron receptor