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Acute lung injury (ALI) represents a continuum of pathologies that causes alveolar epithelial and endothelial cell disruption or dysfunction, which leads to immediate or delayed edema and a significant disturbance in surfactant homeostasis. Acute respiratory distress syndrome (ARDS) is the most severe clinical manifestation of ALI. ARDS is a debilitating and often fatal condition that can result from numerous seemingly unrelated direct or indirect insults. Mortality rates associated with ALI/ARDS have changed little over the last several decades and remain ~30–40%. Because the past candidate-gene approaches have not led to reliable pharmacological treatments, the current treatment strategies primarily depend on supportive measures (e.g., patient repositioning and improved oxygenation strategies) to affect outcome.
The Prows lab has taken a much different approach to study this intractable disease. Because mortality is the most critical endpoint to make an impact, we have used this trait as the endpoint measure in a large-scale genetics approach in mice to identify critical factors affecting differential ALI susceptibility. A genetics approach is not feasible in humans, because ALI/ARDS does not run in families and inciting agents and level of exposures differ among cases. To remove preconceived biases as to which genes or factors are important in survival, we use quantitative trait locus (QTL) analysis—a reverse genetics approach that does not depend on a priori data or information—to steer us towards the responsible genes.
Continuous >95% oxygen (O2; hyperoxia) has been used by research investigators for many years as a prototypical agent to induce ALI in laboratory animals. A screen of a large panel of inbred mice identified C57BL/6J (B) mice as sensitive and 129X1/SvJ (X1) mice as considerably more resistant to the lethal effects of hyperoxia. Genetic analysis of 1,775 recombinant mice, including 840 F2 and 935 backcrosses derived from these strains identified 5 QTLs (i.e. chromosomal regions, designated as Shali1-5) significantly linked to HALI survival time. Mapping results and confidence intervals for the two major loci, Shali1 (chromosome 1) and Shali2 (chromosome 4), are presented in Figure 1. Further analyses revealed that overall survival time also involved decreased penetrance (i.e., the percent of mice that carried the susceptibility gene and expressed the trait) and was significantly affected by sex, cross, and the parent-of-origin.
A closer look at the allelic effects of these two loci in an F2 population derived from X1 and B strain mice revealed opposing actions on survival time. In particular, homozygous X1 alleles were resistant for Shali1, but sensitive for Shali2. Homozygous B alleles were resistant for Shali2, but sensitive for Shali1 (Figure 2). Mice with both sets of sensitive or resistance alleles had significant decreased or increased survival time, providing support for an important gene-gene interaction (Figure 3).
To verify the QTL effects and assess the role of each QTL in HALI survival time, we are constructing reciprocal congenic strains for the 5 Shali regions, a process that involves repeated backcrossing and marker screening of offspring to substitute the QTL region of one strain (donor) with the identical region of another strain (recipient). Figure 4 gives a chromosomal overview of the Shali1 (chromosome 1) and Shali2 (chromosome 4) congenic lines compared to each other and their parental background strains. These congenics have demonstrated dramatic changes in phenotype (Shali1 decreased and Shali2 increased survival time), as compared to the parental X1 strain. Such congenic lines serve as useful models to narrow the region, identify the specific gene(s) involved, and determine the important gene-gene interactions. When refined and validated, these lines will allow detailed mechanistic studies to assess the developing ALI pathology.
Ozone (O3) is a powerful oxidant that can quickly produce an alveolar damage that models the exudative phase of severe ALI. Because O3 is easy to generate and monitor, easy to contain and eliminate, and good data are available on its toxicity and pathology, we have used O3 in genetic studies as a proxy for highly toxic inhaled oxidant gases and a model for chemical-induced ALI (CALI).
In a survey of the most common inbred mouse strains, A/J mice were identified as sensitive and C57BL/6J (B) as highly resistant to O3. Because B mice were sensitive in hyperoxia, but resistant in O3, it suggested that the underlying mechanisms differ for these two oxidants. Separate F2 and backcross populations were generated from the A/J and B progenitors and tested in O3. QTL analysis identified several significant and suggestive QTLs associated with survival time in O3. Though these regions all have potential importance, the main focus has been on Aliq1, a significant region on chromosome 11 that explains ~40% of the survival time difference between A/J and B inbred mice in O3 (Figure 5).
Congenic lines for several of the regions linked to survival time have been constructed, which carry susceptible A/J alleles on the resistant B background. Reciprocal congenics (i.e. B alleles on the A/J background) are also now being generated, for which important regions should demonstrate increased resistance. Future plans are to generate the reciprocal congenics for all significant Aliq loci (as originally identified in the B background). Besides these complex breeding strategies, targeted sequencing, along with combined methods using microarrays, bioinformatics tools and in vivo and in silico studies are planned to identify the causal genes and to characterize their functions. An important question to answer is why the B-strain is highly susceptible to hyperoxic lung injury, but highly resistant to ozone-induced lung injury.
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