Ent system. The lack of CerS2 led to mildly increased lung volumes (Fig. 5B), albeit thePLOS ONE | www.plosone.orgstatic lung compliance was not affected (Fig. 5C). However, we noted a significant increase in airflow resistance in CerS2-null mice compared to WT mice (Fig. 5D). To determine if these functional changes were associated with markers of airway inflammation, we investigated the BAL fluid protein content and cellularity. The BAL fluid from CerS2-null mice exhibited an approximately 25 increase in protein content compared to wild type mice (Fig. 6A) and increased inflammatory cell content. Both the absolute number of alveolar macrophages in the BAL fluid (Fig. 6B), as well as the percentage of lymphocytes and neutrophils were elevated in the airways of CerS2-null mice when compared to wild type mice (Fig.Apocynin 6C).DiscussionWe identified that CerS2 and CerS5 are the most abundant CerS in the lung and CerS2 is necessary for proper lung homeostasis. In particular, CerS2 may be essential for the regulation of lung inflammatory cell homeostasis and the maintenance of the functional integrity of airways and lung airspaces. The molecular mechanisms that account for airway inflammation and increased airflow resistance in CerS2-null mice are not known. The abnormally high C16-ceramide, loss of C24-Sphingolipid Homeostasis Impact on Airway FunctionFigure 4. Effect of CerS2 loss of function on sphingolipid metabolic pathways in the lung. A , 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 6 S.E.M., n = 3; * p,0.05. C , Total lung dihydroceramide levels (C) are increased in CerS2-null mice, paralleled by marked increases in the abundance of C16-dihydroceramide (D); Mean 6 S.Procarbazine Hydrochloride E.PMID:27217159 M., n = 3. 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). doi:10.1371/journal.pone.0062968.gceramides, elevated dihydroceramide or sphinganine levels (unpublished data), or a combination of sphingolipid metabolite accumulation may all be implicated. These in turn, could directly trigger oxidative stress, apoptosis, may alter inflammatory cell trafficking, or alter host-environment interactions. For example, pathological changes in the liver may be caused by chronic oxidative stress, since levels of several anti-oxidant enzymes areelevated, as is lipid peroxidation, protein nitrosylation, and ROS in the liver of CerS2-null mice [16]. The dysmyelinating phenotype of CerS2 deficiency in the brain may be attributed to the reduction in levels of a glycolipid that is enriched in myelin, a conclusion which was based on the high expression levels of CerS2 in oligodendrocytes [17]. In contrast, our investigations of whole lungs and of lung epithelial and endothelial cells indicated similarPLOS ONE | www.plosone.orgSphingolipid Homeostasis Impact on Airway FunctionFigure 5. Lung histology and function of CerS2-null mice. A , 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 , 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 = 54. doi:10.1371/journal.pone.0062968.gPLOS ONE | www.plosone.or.