Figure 1.
Ceramide can be synthesized via the de novo pathway regulated by serine palmitoyl transferase (SPT), ceramide synthases (CerS; isoforms and their preferred substrates described in tabular format), and desaturases (DEGS); via sphingomyelinase pathway regulated by sphingomyelinases (SMases); or via the recycling pathway.
Figure 2.
Ceramide levels and CerS expression in the normal lung and human lung cells.
A, Levels of ceramide species in the whole mouse lung measured by LC-MS/MS (C57BL/6 mice; female; age 3 months; mean+SEM; n = 5). B, Levels of individual CerS mRNA expressed in the whole mouse lung, measured by real time q-rtPCR; mean+SD, n = 5. C–D, Levels of ceramide species (C) and of CerS mRNA (D) in lung structural cells grown in culture: human bronchial epithelial cell line Beas2B, primary human small airway epithelial cells (SAEC), and primary human microvascular endothelial cells (HLMVEC); means+S.E.M., n = 3. Bar colors of ceramide species corresponding to the color of CerS responsible for its synthesis. E, X-Gal staining (blue) of frozen lung sections from CerS2−/+ mice at various magnifications (size bar 100 µm in the left panels and 25 µm in the middle and right panels). Note more prominent transcriptional activity of the LacZ-promoter (blue) in the epithelial layers of the bronchi (b), rather than in the vascular (v) endothelium or alveoli (the arrow indicates an alveolar macrophage).
Figure 3.
Effect of CerS2 loss of function on lung ceramides.
A, Levels of ceramides in the lung of CerS2-null mice (black bars) compared to WT mice (light grey bars) or heterozygous CerS2−/+ mice (dark grey bars). Values are means ± S.E.M., n = 3–5; * p<0.05. B, Total ceramide levels in the lungs of CerS2-null mice (black bars) compared to WT mice (light grey bars); values are means ± S.E.M., n = 3–5. C, Relative expression of ceramide species in WT and CerS2-null mice (percent).
Figure 4.
Effect of CerS2 loss of function on sphingolipid metabolic pathways in the lung.
A–B, Lung acid sphingomyelinase (lysosomal ASM, A) and CerS 5/6 (B) activities in the whole lung are increased in CerS2-null mice compared to wild type. Mean ± S.E.M., n = 3; * p<0.05. C–D, Total lung dihydroceramide levels (C) are increased in CerS2-null mice, paralleled by marked increases in the abundance of C16-dihydroceramide (D); Mean ± S.E.M., n = 3–7. E, Lung C16-ceramide in mice with indicated CerS2 genotype, following treatment with the general CerS inhibitor FB1 or its vehicle, saline (mean+S.E.M., n = 3).
Figure 5.
Lung histology and function of CerS2-null mice.
A–C, Histological changes in the lung parenchyma and bronchi detected by H & E staining of lung sections from CerS2-null and WT mice (A). Note areas of foamy macrophage infiltration (arrowhead), and areas of inflammation. B–D, Lung function measured by lung volumes adjusted by body weight (B), lung compliance (C), and airflow resistance (D) in WT mice or CerS2-null mice; means+S.E.M., n = 5–14.
Figure 6.
Markers of lung remodeling and inflammation in CerS2-null mice lungs.
A–C. Lung inflammation measured by BAL fluid protein content (A, expressed relative to WT control; mean+SEM) and inflammatory cell counts; macrophage numbers (B) and abundance (percent, C) of inflammatory cell macrophages (Mac), lymphocytes (Lym) and polymorphonuclear cells (PMN) in the BAL fluid of WT (light grey) or CerS2-null (black bars) mice, measured by counting on Giemsa-stained cytospin slides; means+S.D., n = 5.