Ambient ozone (O3) exposure has serious consequences on respiratory health, including airway inflammation and injury. Decades of research have yielded thorough descriptions of these outcomes; however, less is known about the molecular processes that drive them. The aim of this study was to further describe the cellular and molecular responses to O3 exposure in murine airways, with a particular focus on transcriptional responses in 2 critical pulmonary tissue compartments: conducting airways (CA) and airway macrophages (AM). After exposing adult, female C57BL/6J mice to filtered air, 1 or 2 ppm O3, we assessed hallmark responses including airway inflammation (cell counts and cytokine secretion) and injury (epithelial permeability), followed by gene expression profiling of CA and AM by RNA-seq. As expected, we observed concentration-dependent increases in airway inflammation and injury. Conducting airways and AM both exhibited changes in gene expression to both 1 and 2 ppm O3 that were largely compartment-specific. In CA, genes associated with epithelial barrier function, detoxification processes, and cellular proliferation were altered, while O3 affected genes involved in innate immune signaling, cytokine production, and extracellular matrix remodeling in AM. Further, CA and AM also exhibited notable differences in concentration–response expression patterns for large numbers of genes. Overall, our study has described transcriptional responses to acute O3 exposure, revealing both shared and unique gene expression patterns across multiple concentrations of O3 and in 2 important O3-responsive tissues. These profiles provide broad mechanistic insight into pulmonary O3 toxicity, and reveal a variety of targets for focused follow-up studies.

Summary17P-Estradiol (0.44 to 4.4 &kg) was intramuscularly administered to pregnant rabbits on day 25 or 26 of gestation, and the fetuses were delivered by cesarean section 24 hr later. On light microscopy, the lungs from the treated group had larger alveoli and thinner interalveolar septa than did those from the controls at the same gestational age. The 1umen:septa ratio was 0.62 + 0.06 in the control group and 0.88 + 0.05 in the treated group ( P < 0.01). Blood vessels in the lungs of the treated group were also more mature than were those in the control group. Alveolar epithelial cells consisted of 52% undifferentiated, 21% type 11, and 27% type I cells in the control group. In the estrogen-treated group, the corresponding distribution was 25, 29, and 45%. There were 0.82 -+ 0.16 lamellar bodies per alveolar cell in the treated group compared to 0.38 k 0.06 in the controls (P < 0.05). Estrogen decreased fetal lung glycogen content from 247 + 15 pg/mg protein to 70 9 on day 26 and from 103 -+ 13 to 13 + 2 on day 27 ( P < 0.001). Estrogen administration increased the rate of incdrporation of choliie into phosphatidylcholine in fetal lung slices. decreased the rate of thymidine incorporation into DNA, but had no effect on the rates bf incorporatibn of ethanolamine into phosphatidylethanolamine or of leucine into protein. These data indicate that estrogen accelerates the rate of fetal lung maturation. It appears to stimulate lung differentiation at the expense of lung growth. SpeculationEstrogen may be involved in the physiologic control of fetal lung maturation and pulmonary surfactant production.We have previously shown that administration of 17/3-estradiol to pregnant rabbits at 25 to 26 days gestation (term is 31 days) accelerates fetal lung maturation and stimulates production of pulmonary surfactant (16,17). Thus, maternal estrogen administration has the following effects on the fetal lung: it increases the amount of total phospholipid and phosphatidylcholine as well as the phosphatidylcholine:sphingomyelin ratio in lung lavage (17), it increases the rate of choline incorporation into phosphatidylcholine in lung slices (16), and it increases the activities of pulmonary cholinephosphate cytidylyltransferase and lysolecithin acyltransferase (16), enzymes involved in the synthesis of total and disaturated phosphatidylcholine, respectively (33). Gross et al. (12) reported that estrogen accelerates maturation of fetal rat lung in organ culture. This fmding suggests that estrogen has a direct effect on the fetal lung.The purpose of the present study was to further characterize the effects of estrogen on fetal lung maturation. Inasmuch as there are distinct morphological changes during maturation of the fetal lung (2), we examined the effects of estrogen on these parameters of lung maturation. In addition, we determined the effect of estrogen on lung glycogen content because this decreases toward the end of gestation in a number of species (4,23,36), and it has been postulated that glycogen may provide substrate o...

Summary MATERIALS A N D M E T H O D Shr. I n the group treated with oxygen plus vitamin E, the lungs were similar to room air controls (82.6% normal). This study thus provides further evidence for a direct antioxident affect of vitamin E in lung. SpeculationThese findings provide further evidence of antioxidant protection of lung by vitamin E and suggest that the antioxidant effect of vitamin E may involve an inactivation of the probable stimulus for antioxidant enzyme induction, oxygen-free radicals.

Summary ated species of this phospholipid is believed to play a major role In lowering surface tension at the air-alveolar interface, thereby PC in the lungs of the fetuses of glucose-intolerant monkeys. InInsulin treatment had no effect on the rate of choline incorpoaddition, Moxley and L~~~~~~~ found ,hat glucose ration into phosphatidylcholine (PC) or disaturated PC and did into PC in isolated, perfused rat lungs was decreased not antagonize the dexamethasone-induced stimulation of choline when the lungs of adult diabetic rats were used. These effects incorporation into PC. When the incorporation of IVilacetate into be reversed by insulin treatment prior to perfusion. the various phospholipid fractions was examined, however, i t was of insulin action. which depend on isotope incorporation into found that exposure to insulin resulted i n a significant decrease i n phospholipids. are, however. complicated by t h e fact insulin the percentage of phospholipid radioactivity in the disaturated PC [he uptake of some substrates into cells and may also fraction and an increase i n the percentage of radioactivity i n the influence the intracellular pool size of these substrates, had the nppnsite effect to insulin with regard to acetate incorpo-phol,p,ds other than PC. was ~h~~~ included the imration into the various phospholipid fractions, and when the two portant surfactant components disaturated PC and phosphatidylhormones were combined, this effect was diminished or abolished. glycerol ( 19. I ) as well as the phosphol,p,dh usually The effects of insulin could not be accounted for on the basis of a with cell membranes. mono-and unsaturated PC.. phosphatidylchange i n activity of any of the enzymes of phospholipid synthesis elhanolamine, phosphat,dylinositol. phosphatidylserine. and that were examined. sphingomyelin (27). The influence of insulin on enzymes involvedThese findings suggest that insulin stimulates the synthesis of with pulmonary phospholipid synthesis was also examined, the cell membrane phospholipids while decreasing that of the surfactant phospholipid, disaturated PC.

Respiratory toxicity caused by the common urban air pollutant ozone (O3) varies considerably within the human population and across inbred mouse strains, suggestive of gene-environment interactions (GxE). Though previous studies genetic mapping studies using classical inbred strains have identified several and quantitative trait locus (QTL) and candidate genes underlying responses to O3 exposure, precise mechanisms of susceptibility remain incompletely described. We sought to expand our understanding of the genetic architecture of O3 responsiveness using the Collaborative Cross (CC) recombinant inbred mouse panel, which contains more genetic diversity than previous inbred strain panels. We evaluated hallmark O3-induced respiratory phenotypes in 56 CC strains after exposure to filtered air or 2 ppm O3, and performed focused genetic analysis of variation in lung injury as measured by the total bronchoalveolar lavage protein concentration. Because animals were exposed in sex- and batch-matched pairs, we defined a protein response phenotype as the difference in lavage protein between the O3- and FA-exposed animal within a pair. The protein response phenotype was heritable, and QTL mapping revealed two novel loci on Chromosomes 10 (peak: 26.2 Mb; 80% CI: 24.6-43.6 Mb) and 15 (peak: 47.1 Mb; 80% CI: 40.2-54.9 Mb), the latter surpassing the 95% significance threshold. At the Chr. 15 locus, C57BL/6J and CAST/EiJ founder haplotypes were associated with higher protein responses compared to all other CC founder strain haplotypes. Using additional statistical analysis and high-density SNP data, we delimited the Chr. 15 QTL to a ~2 Mb region containing 21 genes (10 protein coding). Using a weight of evidence approach that incorporated candidate variant analysis, functional annotations, and publicly available lung gene expression data, we nominated three candidate genes (Oxr1, Rspo2, and Angpt1). In summary, we have shown that O3-induced lung injury is modulated by genetic variation and demonstrated the value of the CC for uncovering and dissecting gene-environment interactions.