Diann M. McCoy

Dynamic regulation of cardiolipin by the lipid pump Atp8b1 determines the severity of lung injury in experimental pneumonia

Pneumonia remains the leading cause of death from infection in the US, yet fundamentally new conceptual models underlying its pathogenesis have not emerged. We show that humans and mice with bacterial pneumonia have markedly elevated amounts of cardiolipin, a rare, mitochondrial-specific phospholipid, in lung fluid and find that it potently disrupts surfactant function. Intratracheal cardiolipin administration in mice recapitulates the clinical phenotype of pneumonia, including impaired lung mechanics, modulation of cell survival and cytokine networks and lung consolidation. We have identified and characterized the activity of a unique cardiolipin transporter, the P-type ATPase transmembrane lipid pump Atp8b1, a mutant version of which is associated with severe pneumonia in humans and mice. Atp8b1 bound and internalized cardiolipin from extracellular fluid via a basic residue–enriched motif. Administration of a peptide encompassing the cardiolipin binding motif or Atp8b1 gene transfer in mice lessened bacteria-induced lung injury and improved survival. The results unveil a new paradigm whereby Atp8b1 is a cardiolipin importer whose capacity to remove cardiolipin from lung fluid is exceeded during inflammation or when Atp8b1 is defective. This discovery opens the door for new therapeutic strategies directed at modulating the abundance or molecular interactions of cardiolipin in pneumonia.

Ceramide Regulates Lipopolysaccharide-Induced Phosphatidylinositol 3-Kinase and Akt Activity in Human Alveolar Macrophages

The phosphatidylinositol (PI) 3-kinase pathway is an important regulator of cell survival. In human alveolar macrophages, we found that LPS activates PI 3-kinase and its downstream effector, Akt. LPS exposure of alveolar macrophages also results in the generation of ceramide. Because ceramide exposure induces apoptosis in other cell types and the PI 3-kinase pathway is known to inhibit apoptosis, we determined the relationship between LPS-induced ceramide and PI 3-kinase activation in alveolar macrophages. We found that ceramide exposure activated PI 3-kinase and Akt. When we blocked LPS-induced ceramide with the inhibitor D609, we blocked LPS-induced PI 3-kinase and Akt activation. Evaluating cell survival after ceramide or LPS exposure, we found that blocking PI 3-kinase induced a significant increase in cell death. Because these effects of PI 3-kinase inhibition were more pronounced in ceramide- vs LPS-treated alveolar macrophages, we also evaluated NF-κB, which has also been linked to cell survival. We found that LPS, to a greater degree than ceramide, induced NF-κB translocation to the nucleus. As a composite, these studies suggest that the effects of ceramide exposure in alveolar macrophages may be very different from the effects described for other cell types. We believe that LPS induction of ceramide results in PI 3-kinase activation and represents a novel effector mechanism that promotes survival of human alveolar macrophages in the setting of pulmonary sepsis.

Fibroblasts Expressing PDGF-Receptor-Alpha Diminish During Alveolar Septal Thinning in Mice

In mice, secondary alveolar septal formation primarily occurs during a brief postnatal period and is accompanied by transient expansion of the interstitial lung fibroblast (LF) population. PDGF-A, which solely signals through PDGF-receptor-alpha (PDGF-Rα), is required for expansion, but the receptors relevant downstream targets remain incompletely defined. We have evaluated the proliferation, apoptosis, and differential response to the selective protein tyrosine kinase inhibitor, imatinib, by pdgfrα-expressing LF (pdgfrα-LF) and compared them with their nonexpressing LF counterparts. Our objective was to determine whether diminished signaling through PDGF-Rα-mediated pathways regulates the decline in myofibroblasts, which accompanies septal thinning and ensures more efficient alveolar gas exchange. Using quantitative stereology and flow cytometry at postnatal d 12 and 14, we observed that imatinib caused a selective suppression of proliferation and an increase in apoptosis. The number of the alpha smooth muscle actin (αSMA) producing pdgfrα-LF was also reduced. Using cultures of neonatal mouse LF, we showed that imatinib did not suppress PDGF-Rα gene expression but reduced PDGF-A-mediated Akt phosphorylation, potentially explaining the increase in apoptosis. Our findings are relevant to bronchopulmonary dysplasia in which positive pressure ventilation interferes with myofibroblast depletion, septal thinning, and capillary maturation.

Identification of Sex-Specific Differences in Surfactant Synthesis in Rat Lung

Delayed lung maturation and lower levels of surfactant phosphatidylcholine have been previously identified in male fetuses compared with female fetuses in several species. We investigated the mechanisms for sex differences in surfactant content by examining parameters of phosphatidylcholine turnover and biosynthesis; the latter was evaluated by measuring metabolic steps within the biosynthetic pathway. Compared with male lung cells, freshly isolated lung cells from female fetuses contained higher levels of disaturated phosphatidylcholine, a marker of surfactant lipid. Female mixed monolayer cultures exhibited a 71% increase in choline incorporation into disaturated phosphatidylcholine compared with male cultures. Male cultures exhibited significantly greater release of [3H]-arachidonic acid into the medium compared with females, suggesting sex differences in phospholipase activity. However, pulse-chase studies showed no sex differences in degradation of disaturated phosphatidylcholine, which was confirmed by assays of phospholipase A2, phosphatidylcholine-specific phospholipase C, and phospholipase D. Female mixed lung cells, however, had greater rates of cellular choline transport and activity of cytidylyltransferase, the rate-regulatory enzyme for phosphatidylcholine synthesis. Separate studies showed that exposure of sex-specific pretype II cell cultures to cortisol-stimulated fibroblast-conditioned medium plus transforming growth factor-β–neutralizing antibody stimulated cytidylyltransferase activity to a greater extent in male cells compared with female cells. These studies indicate that sex differences in surfactant phospholipid content are not due to differences in phospholipid turnover, but rather differential regulation of specific metabolic steps within the surfactant biosynthetic pathway. The data also support a role for transforming growth factor-β as a negative regulator of a key surfactant biosynthetic enzyme within male lungs.

Glucocorticoids Retain Bipotent Fibroblast Progenitors during Alveolar Septation in Mice

&NA; Glucocorticoids have been widely used and exert pleiotropic effects on alveolar structure and function, but do not improve the long‐term clinical outcomes for patients with bronchopulmonary dysplasia, emphysema, or interstitial lung diseases. Treatments that foster alveolar regeneration could substantially improve the long‐term outcomes for such patients. One approach to alveolar regeneration is to stimulate and guide intrinsic alveolar progenitors along developmental pathways used during secondary septation. Other investigators and we have identified platelet‐derived growth factor receptor‐&agr;‐expressing fibroblast subpopulations that are alternatively skewed toward myofibroblast or lipofibroblast phenotypes. In this study, we administered either the glucocorticoid receptor agonist dexamethasone (Dex) or the antagonist mifepristone to mice during the first postnatal week and evaluated their effects on cellular proliferation and adoption of &agr;‐smooth muscle actin and lipid droplets (markers of the myofibroblast and lipofibroblast phenotypes, respectively). We observed that Dex increased the relative abundance of fibroblasts with progenitor characteristics, i.e., containing both &agr;‐smooth muscle actin and lipid droplets, uncoupling protein‐1 (a marker of brown and beige adipocytes), delta‐like ligand‐1, and stem cell antigen‐1. Dex enhanced signaling through the Smad1/5 pathway, which increased uncoupling protein‐1 in a lung fibroblast progenitor cell line. We conclude that glucocorticoid receptor manipulation can sustain fibroblast plasticity, and posit that targeting downstream glucocorticoid responsive pathways could steer fibroblast progenitors along more desirable regenerative pathways.