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 mechanisms of ventilator-induced lung injury, an iatrogenic inflammatory condition induced by mechanical ventilation, are not completely understood. Toll-like receptor 4 (TLR4) signaling via the adaptor protein, myeloid differentiation factor 88 (MyD88) is proinflammatory and plays a critical role in host immune response to invading pathogen and noninfectious tissue injury. The role of TLR4-MyD88 signaling in ventilator-induced lung injury remains incompletely understood.
Mice were ventilated with low or high tidal volume (HTV), 7 or 20 ml/kg, after tracheotomy for 4 h. Control mice were tracheotomized without ventilation. Lung injury was assessed by: alveolar capillary permeability to Evans blue albumin, wet/dry ratio, bronchoalveolar lavage analysis for cell counts, total proteins and cytokines, lung histopathology, and plasma cytokine levels.
Wildtype mice subjected to HTV had increased: pulmonary permeability; inflammatory cell infiltration/lung edema; and interleukin-6/macrophage-inflammatory protein-2 in the lavage compared to control. In HTV, inhibitor of κB alpha decreased whereas phosphorylated extracellular signal-regulated kinases increased. TLR4 mutant and MyD88−/− mice showed markedly attenuated response to HTV, including less lung inflammation; pulmonary edema; and cell number, protein content, and the cytokines in the lavage. Furthermore, compared to wildtype, both TLR4 mutant and MyD88−/− mice had significantly higher inhibitor of κB alpha and reduced extracellular signal-regulated kinases phosphorylation following HTV.
TLR4-MyD88 signaling plays an important role in the development of ventilator-induced lung injury in mice, possibly through mechanisms involving nuclear factor-κB and mitogen-activated protein kinase pathways.
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
Epithelial antimicrobial activity may protect the lung against inhaled pathogens. The bactericidal/permeability-increasing protein family has demonstrated antimicrobial activity in vitro. PLUNC (palate, lung and nasal epithelium carcinoma associated) is a 25 kDa secreted protein that shares homology with bactericidal/permeability-increasing proteins and is expressed in nasopharyngeal and respiratory epithelium. The objective of this study was to determine whether PLUNC can limit Pseudomonas aeruginosa infection in mice. Transgenic mice (Scgb1a1-hPLUNC) were generated in which human PLUNC (hPLUNC) was directed to the airway epithelium with the Scgb1a1 promoter. The human PLUNC protein (hPLUNC) was detected in the epithelium throughout the trachea and bronchial airways and in bronchoalveolar lavage fluid (BALF). BALF from transgenic mice exhibited higher antibacterial activity than that from wild type littermates in vitro. Following in vivo P. aeruginosa challenge, Scgb1a1-hPLUNC transgenic mice displayed enhanced bacterial clearance. This was accompanied by a decrease in neutrophil infiltration and cytokine levels. More importantly, the over-expressed hPLUNC in Scgb1a1-hPLUNC transgenic mouse airway significantly enhanced mouse survival against P. aeruginosa induced respiratory infection. These data indicate that PLUNC is a novel antibacterial protein that likely plays a critical role in airway epithelium mediated innate immune response.
Rationale: Previously, we demonstrated a candidate region for susceptibility to airspace enlargement on mouse chromosome 5. However, the specific candidate genes within this region accounting for emphysema-like changes remain unrecognized. c-Kit is a receptor tyrosine kinase within this candidate gene region that has previously been recognized to contribute to the survival, proliferation, and differentiation of hematopoietic stem cells. Increases in the percentage of cells expressing c-Kit have previously been associated with protection against injury-induced emphysema.
Objectives: Determine whether genetic variants of c-Kit are associated with spontaneous airspace enlargement.
Methods: Perform single-nucleotide polymorphism association studies in the mouse strains at the extremes of airspace enlargement phenotype for variants in c-Kit tyrosine kinase. Characterize mice bearing functional variants of c-Kit compared with wild-type controls for the development of spontaneous airspace enlargement. Epithelial cell proliferation was measured in culture.
Measurements and Main Results: Upstream regulatory single-nucleotide polymorphisms in the divergent mouse strains were associated with the lung compliance difference observed between the extreme strains. c-Kit mutant mice (KitW-sh/W-sh), when compared with genetic controls, developed altered lung histology, increased total lung capacity, increased residual volume, and increased lung compliance that persist into adulthood. c-Kit inhibition with imatinib attenuated in vitro proliferation of cells expressing epithelial cell adhesion molecule.
Conclusions: Our findings indicate that c-Kit sustains and/or maintains normal alveolar architecture in the lungs of mice. In vitro data suggest that c-Kit can regulate epithelial cell clonal expansion. The precise mechanisms that c-Kit contributes to the development of airspace enlargement and increased lung compliance remain unclear and warrants further investigation.
genetic; tyrosine kinase; SASH; chronic obstructive pulmonary disease; aging
Rationale: Because acute lung injury is a sporadic disease produced by heterogeneous precipitating factors, previous genetic analyses are mainly limited to candidate gene case-control studies.
Objectives: To develop a genome-wide strategy in which single nucleotide polymorphism associations are assessed for functional consequences to survival during acute lung injury in mice.
Methods: To identify genes associated with acute lung injury, 40 inbred strains were exposed to acrolein and haplotype association mapping, microarray, and DNA-protein binding were assessed.
Measurements and Main Results: The mean survival time varied among mouse strains with polar strains differing approximately 2.5-fold. Associations were identified on chromosomes 1, 2, 4, 11, and 12. Seven genes (Acvr1, Cacnb4, Ccdc148, Galnt13, Rfwd2, Rpap2, and Tgfbr3) had single nucleotide polymorphism (SNP) associations within the gene. Because SNP associations may encompass “blocks” of associated variants, functional assessment was performed in 91 genes within ± 1 Mbp of each SNP association. Using 10% or greater allelic frequency and 10% or greater phenotype explained as threshold criteria, 16 genes were assessed by microarray and reverse real-time polymerase chain reaction. Microarray revealed several enriched pathways including transforming growth factor-β signaling. Transcripts for Acvr1, Arhgap15, Cacybp, Rfwd2, and Tgfbr3 differed between the strains with exposure and contained SNPs that could eliminate putative transcriptional factor recognition sites. Ccdc148, Fancl, and Tnn had sequence differences that could produce an amino acid substitution. Mycn and Mgat4a had a promoter SNP or 3′untranslated region SNPs, respectively. Several genes were related and encoded receptors (ACVR1, TGFBR3), transcription factors (MYCN, possibly CCDC148), and ubiquitin-proteasome (RFWD2, FANCL, CACYBP) proteins that can modulate cell signaling. An Acvr1 SNP eliminated a putative ELK1 binding site and diminished DNA–protein binding.
Conclusions: Assessment of genetic associations can be strengthened using a genetic/genomic approach. This approach identified several candidate genes, including Acvr1, associated with increased susceptibility to acute lung injury in mice.
acute respiratory distress syndrome; smoke inhalation; carboxyl stress; transforming growth factor-&beta signaling; ubiquitination
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
Acute lung injury can be induced indirectly (e.g., sepsis) or directly (e.g., chlorine inhalation). Because treatment is still limited to supportive measures, mortality remains high (∼74,500 deaths/yr). In the past, accidental (railroad derailments) and intentional (Iraq terrorism) chlorine exposures have led to deaths and hospitalizations from acute lung injury. To better understand the molecular events controlling chlorine-induced acute lung injury, we have developed a functional genomics approach using inbred mice strains. Various mouse strains were exposed to chlorine (45 ppm × 24 h) and survival was monitored. The most divergent strains varied by more than threefold in mean survival time, supporting the likelihood of an underlying genetic basis of susceptibility. These divergent strains are excellent models for additional genetic analysis to identify critical candidate genes controlling chlorine-induced acute lung injury. Gene-targeted mice then could be used to test the functional significance of susceptibility candidate genes, which could be valuable in revealing novel insights into the biology of acute lung injury.
pulmonary edema; vascular permeability; terrorism countermeasures; acute respiratory distress syndrome
In this study we examined the role of the antioxidant glutathione (GSH) in pulmonary susceptibility to ozone toxicity, utilizing GSH deficient C57BL/6J mice that lack the expression of glutamate-cysteine ligase modifier subunit (GCLM). Gclm(−/−) knockout mice had 70% GSH depletion in the lung. Gclm(+/+) wild-type and Gclm(−/−) mice were exposed to either 0.3 ppm ozone or filtered air for 48 hours. Ozone-induced lung hyperpermeability, as measured by total protein concentration in bronchoalveolar lavage fluid, was surprisingly lower in Gclm(−/−) mice than in wild-type mice. Lung hyperpermeability did not correlate with the degree of neutrophilia or with inflammatory gene expression. Pulmonary antioxidant response to ozone, assessed by increased mRNA levels of metallothionein 1 and 2, alpha-tocopherol transporter protein, and solute carrier family 23 member 2 (sodium-dependent vitamin C transporter) was greater in Gclm(−/−) mice than in Gclm(+/+) mice. These results suggest that compensatory augmentation of antioxidant defenses in Gclm(−/−) mice may confer increased resistance to ozone-induced lung injury.
ozone; glutathione; mice; antioxidant genes; lung hyperpermeability
Rationale: Induced mainly by cigarette smoking, chronic obstructive pulmonary disease (COPD) is a global public health problem characterized by progressive difficulty in breathing and increased mucin production. Previously, we reported that acrolein levels found in COPD sputum could activate matrix metalloproteinase-9 (MMP9).
Objectives: To determine whether acrolein increases expression and activity of MMP14, a critical membrane-bound endopeptidase that can initial a MMP-activation cascade.
Methods: MMP14 activity and adduct formation were measured following direct acrolein treatment. MMP14 expression and activity was measured in human airway epithelial cells. MMP14 immunohistochemistry was performed with COPD tissue, and in acrolein- or tobacco-exposed mice.
Measurements and Main Results: In a cell-free system, acrolein, in concentrations equal to those found in COPD sputum, directly adducted cysteine 319 in the MMP14 hemopexin-like domain and activated MMP14. In cells, acrolein increased MMP14 activity, which was inhibited by a proprotein convertase inhibitor, hexa-d-arginine. In the airway epithelium of COPD subjects, immunoreactive MMP14 protein increased. In mouse lung, acrolein or tobacco smoke increased lung MMP14 activity and protein. In cells, acrolein-induced MMP14 transcripts were inhibited by an epidermal growth factor receptor (EGFR) neutralizing antibody, EGFR kinase inhibitor, metalloproteinase inhibitor, or mitogen-activated protein kinase (MAPK) 3/2 or MAPK8 inhibitors, but not a MAPK14 inhibitor. Decreasing the MMP14 protein and activity in vitro by small interfering (si)RNA to MMP14 diminished the acrolein-induced MUC5AC transcripts. In acrolein-exposed mice or transgenic mice with lung-specific transforming growth factor-α (an EGFR ligand) expression, lung MMP14 and MUC5AC levels increased and these effects were inhibited by a EGFR inhibitor, erlotinib.
Conclusions: Taken together, these findings implicate acrolein-induced MMP14 expression and activity in mucin production in COPD.
cigarette smoke; acrolein; erlotinib; mucous cell metaplasia; chronic obstructive pulmonary disease
Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infection in infants, with about half being infected in their first year of life. Yet only 2 to 3% of infants are hospitalized for RSV infection, suggesting that individual susceptibility contributes to disease severity. Previously, we determined that AKR/J (susceptible) mice developed high lung RSV titers and showed delayed weight recovery, whereas C57BL/6J (resistant) mice demonstrated low lung RSV titers and rapid weight recovery. In addition, we have reported that gene-targeted mice lacking the cystic fibrosis transmembrane conductance regulator (Cftr; ATP-binding cassette subfamily C, member 7) are susceptible to RSV infection. For this report, recombinant backcross and F2 progeny derived from C57BL/6J and AKR/J mice were infected with RSV, their lung titers were measured, and quantitative trait locus (QTL) analysis was performed. A major QTL, designated Rsvs1, was identified on proximal mouse chromosome 6 in both recombinant populations. Microarray analysis comparing lung transcripts of the parental strains during infection identified several candidate genes that mapped to the Rsvs1 interval, including Cftr. These findings add to our understanding of individual RSV susceptibility and strongly support a modifier role for CFTR in RSV infection, a significant cause of respiratory morbidity in infants with cystic fibrosis.
Zinc deficiency is common among populations at high risk for sepsis mortality, including elderly, alcoholic, and hospitalized patients. Zinc deficiency causes exaggerated inflammatory responses to endotoxin but has not been evaluated during bacterial sepsis. We hypothesized that subacute zinc deficiency would amplify immune responses and oxidant stress during bacterial sepsis [i.e., cecal ligation and puncture (CLP)] resulting in increased mortality and that acute nutritional repletion of zinc would be beneficial.
Prospective, randomized, controlled animal study.
University medical center research laboratory.
Adult male C57BL/6 mice.
Ten-week-old, male, C57BL/6 mice were randomized into three dietary groups: 1) control diet, 2) zinc-deficient diet for 3 weeks, and 3) zinc-deficient diet for 3 weeks followed by oral zinc supplementation for 3 days (n = 35 per diet). Mice were then assigned to receive either CLP or sham operation (n = 15 each per diet). CLP and sham-operated treatment groups were further assigned to a 7-day survival study (n = 10 per treatment per diet) or were evaluated at 24 hours (n = 5 per treatment per diet) for signs of vital organ damage.
Measurements and Main Results
Sepsis mortality was significantly increased with zinc deficiency (90% vs. 30% on control diet). Zinc-deficient animals subject to CLP had higher plasma cytokines, more severe organ injury, including increased oxidative tissue damage and cell death, particularly in the lungs and spleen. None of the sham-operated animals died or developed signs of organ damage. Zinc supplementation normalized the inflammatory response, greatly diminished tissue damage, and significantly reduced mortality.
Subacute zinc deficiency significantly increases systemic inflammation, organ damage, and mortality in a murine polymicrobial sepsis model. Short-term zinc repletion provides significant, but incomplete protection despite normalization of inflammatory and organ damage indices.
inflammation; cell injury; oxidants; multiple organ dysfunction score; cecal ligation and puncture
Ozone exposure is a growing global health problem, especially in urban areas. While ozone in the stratosphere protects the earth from harmful ultraviolet light, tropospheric or ground-level ozone is toxic and can damage the respiratory tract. It has recently been shown that ozone may be produced endogenously in inflammation and antibacterial responses of the immune system; however, these results have sparked controversy owing to the use of a non-specific colorimetric probe. Here we report the synthesis of fluorescent molecular probes able to unambiguously detect ozone in both biological and atmospheric samples. Unlike other ozone-detection methods, in which interference from different reactive oxygen species is often a problem, these probes are ozone specific. Such probes will prove useful for the study of ozone in environmental science and biology, and so possibly provide some insight into the role of ozone in cells.
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
Motivation: The elucidation of biological pathways enriched with differentially expressed genes has become an integral part of the analysis and interpretation of microarray data. Several statistical methods are commonly used in this context, but the question of the optimal approach has still not been resolved.
Results: We present a logistic regression-based method (LRpath) for identifying predefined sets of biologically related genes enriched with (or depleted of) differentially expressed transcripts in microarray experiments. We functionally relate the odds of gene set membership with the significance of differential expression, and calculate adjusted P-values as a measure of statistical significance. The new approach is compared with Fisher's exact test and other relevant methods in a simulation study and in the analysis of two breast cancer datasets. Overall results were concordant between the simulation study and the experimental data analysis, and provide useful information to investigators seeking to choose the appropriate method. LRpath displayed robust behavior and improved statistical power compared with tested alternatives. It is applicable in experiments involving two or more sample types, and accepts significance statistics of the investigator's choice as input.
Availability: An R function implementing LRpath can be downloaded from http://eh3.uc.edu/lrpath.
Supplementary information: Supplementary data are available at Bioinformatics online and at http://eh3.uc.edu/lrpath.
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
Persistent macrophage accumulation and alveolar enlargement are hallmark features of chronic obstructive pulmonary disease (COPD). A role for CD8+ lymphocytes in the development of COPD is suggested based on observations that this T cell subset is increased in the airways and parenchyma of smokers that develop COPD with airflow limitation. In this study, we utilize a mouse model of COPD to examine the contributions of CD8+ T cells in the persistent macrophage accumulation and airspace enlargement resulting from chronic irritant exposure.
We analyzed pulmonary inflammation and alveolar destruction in wild-type and Cd8-deficient mice chronically exposed to acrolein, a potent respiratory tract irritant. We further examined cytokine mRNA expression levels by RNase protection assay, matrix metalloproteinase (MMP) activity by gelatin zymography, and epithelial cell apoptosis by active caspase3 immunohistochemistry in wild-type and Cd8-deficient mice exposed chronically to acrolein.
These studies demonstrate that CD8+ T cells, are important mediators of macrophage accumulation in the lung and the progressive airspace enlargement in response to chronic acrolein exposures. The expression of several inflammatory cytokines (IP-10, IFN-γ, IL-12, RANTES, and MCP-1), MMP2 and MMP9 gelatinase activity, and caspase3 immunoreactivity in pulmonary epithelial cells were attenuated in the Cd8-deficient mice compared to wild-type.
These results indicate that CD8+ T cells actively contribute to macrophage accumulation and the development of irritant-induced airspace enlargement.
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
The small sample sizes often used for microarray experiments result in poor estimates of variance if each gene is considered independently. Yet accurately estimating variability of gene expression measurements in microarray experiments is essential for correctly identifying differentially expressed genes. Several recently developed methods for testing differential expression of genes utilize hierarchical Bayesian models to "pool" information from multiple genes. We have developed a statistical testing procedure that further improves upon current methods by incorporating the well-documented relationship between the absolute gene expression level and the variance of gene expression measurements into the general empirical Bayes framework.
We present a novel Bayesian moderated-T, which we show to perform favorably in simulations, with two real, dual-channel microarray experiments and in two controlled single-channel experiments. In simulations, the new method achieved greater power while correctly estimating the true proportion of false positives, and in the analysis of two publicly-available "spike-in" experiments, the new method performed favorably compared to all tested alternatives. We also applied our method to two experimental datasets and discuss the additional biological insights as revealed by our method in contrast to the others. The R-source code for implementing our algorithm is freely available at .
We use a Bayesian hierarchical normal model to define a novel Intensity-Based Moderated T-statistic (IBMT). The method is completely data-dependent using empirical Bayes philosophy to estimate hyperparameters, and thus does not require specification of any free parameters. IBMT has the strength of balancing two important factors in the analysis of microarray data: the degree of independence of variances relative to the degree of identity (i.e. t-tests vs. equal variance assumption), and the relationship between variance and signal intensity. When this variance-intensity relationship is weak or does not exist, IBMT reduces to a previously described moderated t-statistic. Furthermore, our method may be directly applied to any array platform and experimental design. Together, these properties show IBMT to be a valuable option in the analysis of virtually any microarray experiment.
Rationale: Acute lung injury can occur from multiple causes, resulting in high mortality. The pathophysiology of nickel-induced acute lung injury in mice is remarkably complex, and the molecular mechanisms are uncertain.
Objectives: To integrate molecular pathways and investigate the role of transforming growth factor β (TGF-β) in acute lung injury in mice.
Methods: cDNA microarray analyses were used to identify lung gene expression changes after nickel exposure. MAPPFinder analysis of the microarray data was used to determine significantly altered molecular pathways. TGF-β1 protein in bronchoalveolar lavage fluid, as well as the effect of inhibition of TGF-β, was assessed in nickel-exposed mice. The effect of TGF-β on surfactant-associated protein B (Sftpb) promoter activity was measured in mouse lung epithelial cells.
Measurements and Main Results: Genes that decreased the most after nickel exposure play important roles in lung fluid absorption or surfactant and phospholipid synthesis, and genes that increased the most were involved in TGF-β signaling. MAPPFinder analysis further established TGF-β signaling to be significantly altered. TGF-β–inducible genes involved in the regulation of extracellular matrix function and fibrinolysis were significantly increased after nickel exposure, and TGF-β1 protein was also increased in the lavage fluid. Pharmacologic inhibition of TGF-β attenuated nickel-induced protein in bronchoalveolar lavage. In addition, treatment with TGF-β1 dose-dependently repressed Sftpb promoter activity in vitro, and a novel TGF-β–responsive region in the Sftpb promoter was identified.
Conclusions: These data suggest that TGF-β acts as a central mediator of acute lung injury through the alteration of several different molecular pathways.
microarray; surfactant; fibrinolysis; extracellular matrix
Asthma has a high prevalence in the United States, and persons with asthma may be at added risk from the adverse effects of hazardous air pollutants (HAPs). Complex mixtures (fine particulate matter and tobacco smoke) have been associated with respiratory symptoms and hospital admissions for asthma. The toxic ingredients of these mixtures are HAPs, but whether ambient HAP exposures can induce asthma remains unclear. Certain HAPs are occupational asthmagens, whereas others may act as adjuncts during sensitization. HAPs may exacerbate asthma because, once sensitized, individuals can respond to remarkably low concentrations, and irritants lower the bronchoconstrictive threshold to respiratory antigens. Adverse responses after ambient exposures to complex mixtures often occur at concentrations below those producing effects in controlled human exposures to a single compound. In addition, certain HAPs that have been associated with asthma in occupational settings may interact with criteria pollutants in ambient air to exacerbate asthma. Based on these observations and past experience with 188 HAPs, a list of 19 compounds that could have the highest impact on the induction or exacerbation of asthma was developed. Nine additional compounds were identified that might exacerbate asthma based on their irritancy, respirability, or ability to react with biological macromolecules. Although the ambient levels of these 28 compounds are largely unknown, estimated exposures from emissions inventories and limited air monitoring suggest that aldehydes (especially acrolein and formaldehyde) and metals (especially nickel and chromium compounds) may have possible health risk indices sufficient for additional attention. Recommendations for research are presented regarding exposure monitoring and evaluation of biologic mechanisms controlling how these substances induce and exacerbate asthma.