Gpr63 is a modifier of microcephaly in Ttc21b mouse mutants.

The primary cilium is a signaling center critical for proper embryonic development. Previous studies have demonstrated that mice lacking Ttc21b have impaired retrograde trafficking within the cilium and multiple organogenesis phenotypes, including microcephaly. Interestingly, the severity of the microcephaly in Ttc21baln/aln homozygous null mutants is considerably affected by the genetic background and mutants on an FVB/NJ (FVB) background develop a forebrain significantly smaller than mutants on a C57BL/6J (B6) background. We performed a Quantitative Trait Locus (QTL) analysis to identify potential genetic modifiers and identified two regions linked to differential forebrain size: modifier of alien QTL1 (Moaq1) on chromosome 4 at 27.8 Mb and Moaq2 on chromosome 6 at 93.6 Mb. These QTLs were validated by constructing congenic strains. Further analysis of Moaq1 identified an orphan G-protein coupled receptor (GPCR), Gpr63, as a candidate gene. We identified a SNP that is polymorphic between the FVB and B6 strains in Gpr63 and creates a missense mutation predicted to be deleterious in the FVB protein. We used CRISPR-Cas9 genome editing to create two lines of FVB congenic mice: one with the B6 sequence of Gpr63 and the other with a deletion allele leading to a truncation of the GPR63 C-terminal tail. We then demonstrated that Gpr63 can localize to the cilium in vitro. These alleles affect ciliary localization of GPR63 in vitro and genetically interact with Ttc21baln/aln as Gpr63;Ttc21b double mutants show unique phenotypes including spina bifida aperta and earlier embryonic lethality. This validated Gpr63 as a modifier of multiple Ttc21b neural phenotypes and strongly supports Gpr63 as a causal gene (i.e., a quantitative trait gene, QTG) within the Moaq1 QTL.

Genetic determinants of ammonia-induced acute lung injury in mice.

In this study, a genetically diverse panel of 43 mouse strains was exposed to ammonia, and genome-wide association mapping was performed employing a single-nucleotide polymorphism (SNP) assembly. Transcriptomic analysis was used to help resolve the genetic determinants of ammonia-induced acute lung injury. The encoded proteins were prioritized based on molecular function, nonsynonymous SNP within a functional domain or SNP within the promoter region that altered expression. This integrative functional approach revealed 14 candidate genes that included Aatf, Avil, Cep162, Hrh4, Lama3, Plcb4, and Ube2cbp, which had significant SNP associations, and Aff1, Bcar3, Cntn4, Kcnq5, Prdm10, Ptcd3, and Snx19, which had suggestive SNP associations. Of these genes, Bcar3, Cep162, Hrh4, Kcnq5, and Lama3 are particularly noteworthy and had pathophysiological roles that could be associated with acute lung injury in several ways.

Heart disease in a mutant mouse model of spontaneous eosinophilic myocarditis maps to three loci.

BACKGROUND: Heart disease (HD) is the major cause of morbidity and mortality in patients with hypereosinophilic diseases. Due to a lack of adequate animal models, our understanding of the pathophysiology of eosinophil-mediated diseases with heart complications is limited. We have discovered a mouse mutant, now maintained on an A/J inbred background, that spontaneously develops hypereosinophilia in multiple organs. Cellular infiltration into the heart causes an eosinophilic myocarditis, with affected mice of the mutant line (i.e., A/JHD) demonstrating extensive myocardial damage and remodeling that leads to HD and premature death, usually by 15-weeks old. RESULTS: Maintaining the A/JHD line for many generations established that the HD trait was heritable and implied the mode of inheritance was not too complex. Backcross and intercross populations generated from mating A/JHD males with females from four different inbred strains produced recombinant populations with highly variable rates of affected offspring, ranging from none in C57BL/6 J intercrosses, to a few mice with HD using 129S1/SvImJ intercrosses and C57BL/6 J backcrosses, but nearly 8% of intercrosses and > 17% of backcrosses from SJL/J related populations developed HD. Linkage analyses of these SJL/J derived recombinants identified three highly significant loci: a recessive locus mapping to distal chromosome 5 (LOD = 4.88; named Emhd1 for eosinophilic myocarditis to heart disease-1); and two dominant variants mapping to chromosome 17, one (Emhd2; LOD = 7.51) proximal to the major histocompatibility complex, and a second (Emhd3; LOD = 6.89) that includes the major histocompatibility region. Haplotype analysis identified the specific crossovers that defined the Emhd1 (2.65 Mb), Emhd2 (8.46 Mb) and Emhd3 (14.59 Mb) intervals. CONCLUSIONS: These results indicate the HD trait in this mutant mouse model of eosinophilic myocarditis is oligogenic with variable penetrance, due to multiple segregating variants and possibly additional genetic or nongenetic factors. The A/JHD mouse model represents a unique and valuable resource to understand the interplay of causal factors that underlie the pathology of this newly discovered eosinophil-associated disease with cardiac complications.

Characterization of a mouse model of hypereosinophilia-associated heart disease.

Hypereosinophilic syndrome is characterized by sustained and marked eosinophilia leading to tissue damage and organ dysfunction. Morbidity and mortality occur primarily due to cardiac and thromboembolic complications. Understanding the cause and mechanism of disease would aid in the development of targeted therapies with greater efficacy and fewer side effects. We discovered a spontaneous mouse mutant in our colony with a hypereosinophilic phenotype. Mice develop peripheral blood eosinophilia; infiltration of lungs, spleen, and heart by eosinophils; and extensive myocardial damage and remodeling. This ultimately leads to heart failure and premature death. Histopathological assessment of the hearts revealed a robust inflammatory infiltrate composed primarily of eosinophils and B-lymphocytes, associated with myocardial damage and replacement fibrosis, consistent with eosinophilic myocarditis. In many cases, hearts showed dilatation and thinning of the right ventricular wall, suggestive of an inflammatory dilated cardiomyopathy. Most mice showed atrial thrombi, which often filled the chamber. Protein expression analysis revealed overexpression of chemokines and cytokines involved in innate and adaptive immunity including IL-4, eotaxin, and RANTES. Disease could be transferred to wild-type mice by adoptive transfer of splenocytes from affected mice, suggesting a role for the immune system. In summary, the pathologies observed in the mutant lines are reminiscent of those seen in patients with hypereosinophilia, where cardiac-related morbidities, like congestive heart failure and thrombi, are the most common causes of death. As such, our model provides an opportunity to test mechanistic hypotheses and develop targeted therapies.NEW & NOTEWORTHY This article describes a new model of heart disease in hypereosinophilia. The model developed as a spontaneous mouse mutant in the colony and is characterized by peripheral blood eosinophilia and infiltration of lungs, spleen, and heart by eosinophils. In the heart, there is extensive myocardial damage, remodeling, fibrosis, and thrombosis, leading to heart failure and death. The immune microenvironment is one of increased innate and adaptive immunity, including Th1 and Th2 cytokines/chemokines. Finally, adoptive transfer of splenocytes transfers disease to recipient mice. In summary, this model provides an opportunity to test mechanistic hypotheses and develop targeted therapies for this rare but devastating disease.

Genetic susceptibility to toxicologic lung responses among inbred mouse strains following exposure to carbon nanotubes and profiling of underlying gene networks.

The risk of human exposure to fiber nanoparticles has risen in recent years due to increases in the manufacture and utilization of carbon nanotubes (CNTs). CNTs are present as airborne particulates in occupational settings and their hazard potential has been demonstrated in experimental lung exposure studies using inbred mouse strains. However, it is not known whether different inbred strains differ in lung responses to CNTs by virtue of their genetics. In this work, common inbred strains (BALB/c, C57Bl/6, DBA/2, and C3H/He) were exposed to CNTs via oropharyngeal aspiration and lung histology and bronchoalveolar lavage (BAL) samples were evaluated over 28days with the objective of evaluating sensitivity/resistance among strains. C57Bl/6 mice developed significantly more extensive type II pneumocyte (T2P) hyperplasia and alveolar infiltrate compared to DBA/2 mice, which were resistant. Surprisingly, DBA/2 but not C57Bl/6 mice were extremely sensitive to increases in leukocytes recovered in BAL fluid. Underlying global gene expression patterns in the two strains were compared using mRNA sequencing to investigate regulatory networks associated with the different effects. The impact of exposure on gene networks regulating various aspects of immune response and cell survival was limited in DBA/2 mice compared to C57Bl/6. Investigation of B6D2F1 (C57Bl/6×DBA/2 hybrid) mice demonstrated inheritance of sensitivity to CNT exposures in regard to toxicologic lung pathology and BAL leukocyte accumulations. These findings demonstrate a genetic basis of susceptibility to CNT particle exposures and both inform the use of inbred mouse models and suggest the likelihood of differences in genetic susceptibility among humans.

A computational framework for the inheritance pattern of genomic imprinting for complex traits.

Genetic imprinting, by which the expression of a gene depends on the parental origin of its alleles, may be subjected to reprogramming through each generation. Currently, such reprogramming is limited to qualitative description only, lacking more precise quantitative estimation for its extent, pattern and mechanism. Here, we present a computational framework for analyzing the magnitude of genetic imprinting and its transgenerational inheritance mode. This quantitative model is based on the breeding scheme of reciprocal backcrosses between reciprocal F(1) hybrids and original inbred parents, in which the transmission of genetic imprinting across generations can be tracked. We define a series of quantitative genetic parameters that describe the extent and transmission mode of genetic imprinting and further estimate and test these parameters within a genetic mapping framework using a new powerful computational algorithm. The model and algorithm described will enable geneticists to identify and map imprinted quantitative trait loci and dictate a comprehensive atlas of developmental and epigenetic mechanisms related to genetic imprinting. We illustrate the new discovery of the role of genetic imprinting in regulating hyperoxic acute lung injury survival time using a mouse reciprocal backcross design.

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

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 ß1 signaling, which previously has been associated with the susceptibility to ALI in mice.

Integrative assessment of chlorine-induced acute lung injury in mice.

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.

Characterization of genomic imprinting effects and patterns with parametric accelerated failure time model.

Genomic imprinting, a genetic phenomenon of non-equivalent allele expression that depends on parental origins, has been ubiquitously observed in nature. It does not only control the traits of growth and development but also may be responsible for survival traits. Based on the accelerated failure time model, we construct a general parametric model for mapping the imprinted QTL (iQTL). Within the framework of interval mapping, maximum likelihood estimation of iQTL parameters is implemented via EM algorithm. The imprinting patterns of the detected iQTL are statistically tested according to a series of null hypotheses. BIC model selection criterion is employed to choose an optimal baseline hazard function with maximum likelihood and parsimonious parameters. Simulations are used to validate the proposed mapping procedure. A published dataset from a mouse model system was used to illustrate the proposed framework. Results show that among the five commonly used survival distributions, Log-logistic distribution is the optimal baseline hazard function for mapping QTL of hyperoxic acute lung injury (HALI) survival; under the log-logistic distribution, four QTLs were identified, in which only one QTL was inherited in Mendelian fashion, whereas others were imprinted in different imprinting patterns.

Haplotype association mapping of acute lung injury in mice implicates activin a receptor, type 1.

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-ß 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.

Generalized F accelerated failure time model for mapping survival trait loci.

As the two most popular models in survival analysis, the accelerated failure time (AFT) model can more easily fit survival data than the Cox proportional hazards model (PHM). In this study, we develop a general parametric AFT model for identifying survival trait loci, in which the flexible generalized F distribution, including many commonly used distributions as special cases, is specified as the baseline survival distribution. EM algorithm for maximum likelihood estimation of model parameters is given. Simulations are conducted to validate the flexibility and the utility of the proposed mapping procedure. In analyzing survival time following hyperoxic acute lung injury (HALI) of mice in an F(2) mating population, the generalized F distribution performed best among the six competing survival distributions and detected four QTLs controlling differential HALI survival.

Multigenic control and sex bias in host susceptibility to spore-induced pulmonary anthrax in mice.

Mechanisms underlying susceptibility to anthrax infection are unknown. Using a phylogenetically diverse panel of inbred mice and spores of Bacillus anthracis Ames, we investigated host susceptibility to pulmonary anthrax. Susceptibility profiles for survival time and organ pathogen load differed across strains, indicating distinct genetic controls. Tissue infection kinetics analysis showed greater systemic dissemination in susceptible DBA/2J (D) mice but a higher terminal bacterial load in resistant BALB/cJ (C) mice. Interestingly, the most resistant strains, C and C57BL/6J (B), demonstrated a sex bias for susceptibility. For example, BALB/cJ females had a significantly higher survival time and required 4-fold more spores for 100% mortality compared to BALB/cJ males. To identify genetic regions associated with differential susceptibility, survival time and extent of organ infection were assessed using mice derived from two susceptibility models: (i) BXD advanced recombinant inbred strains and (ii) F2 offspring generated from polar responding C and D strains. Genome-wide analysis of BXD strain survival identified linkage on chromosomes 5, 6, 9, 11, and 14. Quantitative trait locus (QTL) analysis of the C×DF2 population revealed a significant QTL (designated Rpai1 for resistance to pulmonary anthrax infection, locus 1) for survival time on chromosome 17 and also identified a chromosome 11 locus for lung pathogen burden. The striking difference between genome-wide linkage profiles for these two mouse models of anthrax susceptibility supports our hypothesis that these are multigenic traits. Our data provide the first evidence for a differential sex response to anthrax resistance and further highlight the unlikelihood of a single common genetic contribution for this response across strains.

Genomewide association analysis of respiratory syncytial virus infection in mice.

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.

Bayesian model selection for characterizing genomic imprinting effects and patterns.

MOTIVATION: Although imprinted genes have been ubiquitously observed in nature, statistical methodology still has not been systematically developed for jointly characterizing genomic imprinting effects and patterns. To detect imprinting genes influencing quantitative traits, the least square and maximum likelihood approaches for fitting a single quantitative trait loci (QTL) and Bayesian method for simultaneously modeling multiple QTLs have been adopted in various studies. RESULTS: In a widely used F(2) reciprocal mating population for mapping imprinting genes, we herein propose a genomic imprinting model which describes additive, dominance and imprinting effects of multiple imprinted quantitative trait loci (iQTL) for traits of interest. Depending upon the estimates of the above genetic effects, we categorized imprinting patterns into seven types, which provides a complete classification scheme for describing imprinting patterns. Bayesian model selection was employed to identify iQTL along with many genetic parameters in a computationally efficient manner. To make statistical inference on the imprinting types of iQTL detected, a set of Bayes factors were formulated using the posterior probabilities for the genetic effects being compared. We demonstrated the performance of the proposed method by computer simulation experiments and then applied this method to two real datasets. Our approach can be generally used to identify inheritance modes and determine the contribution of major genes for quantitative variations.

Statistical optimization of parametric accelerated failure time model for mapping survival trait loci.

Most existing statistical methods for mapping quantitative trait loci (QTL) are not suitable for analyzing survival traits with a skewed distribution and censoring mechanism. As a result, researchers incorporate parametric and semi-parametric models of survival analysis into the framework of the interval mapping for QTL controlling survival traits. In survival analysis, accelerated failure time (AFT) model is considered as a de facto standard and fundamental model for data analysis. Based on AFT model, we propose a parametric approach for mapping survival traits using the EM algorithm to obtain the maximum likelihood estimates of the parameters. Also, with Bayesian information criterion (BIC) as a model selection criterion, an optimal mapping model is constructed by choosing specific error distributions with maximum likelihood and parsimonious parameters. Two real datasets were analyzed by our proposed method for illustration. The results show that among the five commonly used survival distributions, Weibull distribution is the optimal survival function for mapping of heading time in rice, while Log-logistic distribution is the optimal one for hyperoxic acute lung injury.

Surfactant-associated protein B is critical to survival in nickel-induced injury in mice.

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 alpha (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.

Reciprocal backcross mice confirm major loci linked to hyperoxic acute lung injury survival time.

Morbidity and mortality associated with acute lung injury (ALI) and acute respiratory distress syndrome remain substantial. Although many candidate genes have been tested, a clear understanding of the pathogenesis is lacking, as is our ability to predict individual outcome. Because ALI is a complex disease, single gene approaches cannot easily identify effectors that must be treated concurrently. We employed a strategy to help identify critical genes and gene combinations involved in ALI mortality. Using hyperoxia to induce ALI, a mouse model for genetic analyses of ALI survival time was identified: C57BL/6J (B) mice are sensitive (i.e., die early), whereas 129X1/SvJ (S) mice are significantly more resistant, but with low penetrance. Segregation analysis of reciprocal F(2) mice generated from B and S strains revealed significant sex, cross, and parent of origin effects. Quantitative trait locus (QTL) analysis identified five chromosomal regions significantly linked to hyperoxic ALI survival time (named Shali1-Shali5). Further analyses demonstrated that both parental strains contribute resistance alleles to their offspring and that the phenotype demonstrated parent of origin effects. To validate earlier findings, we generated and tested mice from all eight possible B-S-derived backcrosses. Results from segregation and QTL analyses of 935 backcrosses, alone and combined with the previous 840 B-S-derived F(2) population, further supported the highly significant QTLs on chromosomes 1 (Shali1) and 4 (Shali2) and confirmed that the sex, cross, and parent of origin all contribute to survival time with hyperoxic ALI.

Surfactant protein C-deficient mice are susceptible to respiratory syncytial virus infection.

Patients with mutations in the pulmonary surfactant protein C (SP-C) gene develop interstitial lung disease and pulmonary exacerbations associated with viral infections including respiratory syncytial virus (RSV). Pulmonary infection with RSV caused more severe interstitial thickening, air space consolidation, and goblet cell hyperplasia in SP-C-deficient (Sftpc(-/-)) mice compared with SP-C replete mice. The RSV-induced pathology resolved more slowly in Sftpc(-/-) mice with lung inflammation persistent up to 30 days postinfection. Polymorphonuclear leukocyte and macrophage counts were increased in the bronchoalveolar lavage (BAL) fluid of Sftpc(-/-) mice. Viral titers and viral F and G protein mRNA were significantly increased in both Sftpc(-/-) and heterozygous Sftpc(+/-) mice compared with controls. Expression of Toll-like receptor 3 (TLR3) mRNA was increased in the lungs of Sftpc(-/-) mice relative to Sftpc(+/+) mice before and after RSV infection. Consistent with the increased TLR3 expression, BAL inflammatory cells were increased in the Sftpc(-/-) mice after exposure to a TLR3-specific ligand, poly(I:C). Preparations of purified SP-C and synthetic phospholipids blocked poly(I:C)-induced TLR3 signaling in vitro. SP-C deficiency increases the severity of RSV-induced pulmonary inflammation through regulation of TLR3 signaling.

Reciprocal congenic lines of mice capture the aliq1 effect on acute lung injury survival time.

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 F(2) 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.

A genetic mouse model to investigate hyperoxic acute lung injury survival.

Acute lung injury (ALI) is a devastating disease that maintains a high mortality rate, despite decades of research. Hyperoxia, a universal treatment for ALI and other critically ill patients, can itself cause pulmonary damage, which drastically restricts its therapeutic potential. We stipulate that having the ability to use higher levels of supplemental O2 for longer periods would improve recovery rates. Toward this goal, a mouse model was sought to identify genes contributing to hyperoxic ALI (HALI) mortality. Eighteen inbred mouse strains were screened in continuous >95% O2. A significant survival difference was identified between sensitive C57BL/6J and resistant 129X1/SvJ strains. Although resistant, only one-fourth of 129X1/SvJ mice survived longer than any C57BL/6J mouse, demonstrating decreased penetrance of resistance. A survival time difference between reciprocal F1 mice implicated a parent-of-origin (imprinting) effect. To further evaluate imprinting and begin to delineate the genetic components of HALI survival, we generated and phenotyped offspring from all four possible intercrosses. Segregation analysis supported maternal inheritance of one or more genes but paternal inheritance of one or more contributor genes. A significant sex effect was demonstrated, with males more resistant than females for all F2 crosses. Survival time ranges and sensitive-to-resistant ratios of the different F2 crosses also supported imprinting and predicted that increased survival is due to dominant resistance alleles contributed by both the resistant and sensitive parental strains. HALI survival is multigenic with a complex mode of inheritance, which should be amenable to genetic dissection with this mouse model.