Ng with hematoxylin and eosin.PLOS 1 | plosone.orgSphingolipid Homeostasis Impact on Airway FunctionFigure 2. Ceramide levels and CerS expression within the standard lung and human lung cells. A, Levels of ceramide species in the complete mouse lung measured by LC-MS/MS (C57BL/6 mice; female; age three months; mean+SEM; n = 5). B, Levels of person CerS mRNA expressed in the complete mouse lung, measured by real time q-rtPCR; mean+SD, n = five. C , Levels of ceramide species (C) and of CerS mRNA (D) in lung structural cells grown in culture: human bronchial epithelial cell line Beas2B, main human compact airway epithelial cells (SAEC), and principal human microvascular endothelial cells (HLMVEC); means+S.E.M., n = three. Bar colors of ceramide species corresponding towards the color of CerS accountable for its synthesis. E, XGal staining (blue) of frozen lung sections from CerS22/+ mice at different magnifications (size bar 100 mm in the left panels and 25 mm within the middle and right panels). Note more prominent transcriptional activity in the LacZ-promoter (blue) inside the epithelial layers with the bronchi (b), in lieu of in the vascular (v) endothelium or alveoli (the arrow indicates an alveolar macrophage). doi:10.1371/journal.pone.0062968.gmultiple effects on ceramide metabolism in lung, equivalent to those observed in other tissues when CerS2 was inhibited. To understand the influence of CerS2 dysregulation around the lung ceramide homeostasis, we measured ceramide metabolites or enzymes within the sphingolipid pathway within the lungs of CerS2-nullmice. The acid sphingomyelinase activity was elevated (Fig. 4A), whereas we could detect no increases within the neutral sphingomyelinase activity (data not shown). Lung ceramide synthase 5/6 activity, accountable for C16-ceramide synthesis was also enhanced (Fig. 4B), suggesting the de novo and/or recycling pathways werePLOS One | plosone.orgSphingolipid Homeostasis Effect on Airway FunctionFigure three. Effect of CerS2 loss of function on lung ceramides. A, Levels of ceramides inside the lung of CerS2-null mice (black bars) compared to WT mice (light grey bars) or heterozygous CerS22/+ mice (dark grey bars). Values are signifies six S.E.M., n = three?; * p,0.05. B, Total ceramide levels within the lungs of CerS2-null mice (black bars) in comparison to WT mice (light grey bars); values are suggests 6 S.E.M., n = 3?. C, Relative expression of ceramide species in WT and CerS2-null mice (%). doi:ten.1371/journal.pone.0062968.Azido-PEG1 supplier galso stimulated in CerS2-null lungs.1219741-19-1 Price These alterations have been associated with greater dihydroceramide levels in the lungs of CerS2-null than in WT mice (Fig.PMID:25023702 4C), mostly on account of C16-dihydroceramide (Fig. 4D). These information pointed to an increase of de novo C16-ceramide synthesis, in parallel to that generated by sphingomyelinase hydrolysis. The increases in C16-ceramide and its precursor have been noted all through postnatal lung development (data not shown). To investigate the contribution of CerS5/6 to C16-ceramide levels in CerS2-null mice, we administered a general CerS inhibitor, FB1. Following three days of systemic FB1 administration, lung C16-ceramide levels substantially declined by approximately 30 , implicating a compensatory upregulation with the de novo pathway of ceramide synthesis in transgenic mice devoid of VLC ceramides (Fig. 4E). To ascertain the part of CerS2 on lung structure and function, lung histology was examined by hematoxylin-eosin staining of lungs inflated beneath constant stress (Fig. 5A ). Lungs from adult mice (3 months or.