Lipidomics reveals dramatic lipid compositional changes in the maturing postnatal lung

Abstract

Lung immaturity is a major cause of morbidity and mortality in premature infants. Understanding the molecular mechanisms driving normal lung development could provide insights on how to ameliorate disrupted development. While transcriptomic and proteomic analyses of normal lung development have been previously reported, characterization of changes in the lipidome is lacking. Lipids play significant roles in the lung, such as dipalmitoylphosphatidylcholine in pulmonary surfactant; however, many of the roles of specific lipid species in normal lung development, as well as in disease states, are not well defined. In this study, we used liquid chromatography-mass spectrometry (LC-MS/MS) to investigate the murine lipidome during normal postnatal lung development. Lipidomics analysis of lungs from post-natal day 7, day 14 and 6-8 week mice (adult) identified 924 unique lipids across 21 lipid subclasses, with dramatic alterations in the lipidome across developmental stages. Our data confirmed previously recognized aspects of post-natal lung development and revealed several insights, including in sphingolipid-mediated apoptosis, inflammation and energy storage/usage. Complementary proteomics, metabolomics and chemical imaging corroborated these observations. This multi-omic view provides a unique resource and deeper insight into normal pulmonary development.

Figure 1. Summary of total lipid identifications color-coded by lipid category and subclass.

The majority of lipids identified belonged to the lipid category glycerophospholipid (PL, blue), with the second most belonging to glycerolipid (GL, purple), and the least identifications belonging to sphingolipids (SP, green). The highest number of identifications by lipid subclass belonged to triacylglycerols (TG, 253 identifications), diacylglycerophosphocholines (PC, 128), and diacylglycerophosphoglycerols (PG, 105).

Figure 2. MCSFA are found at highest abundance in PND7 and PND14 murine lungs as compared to adult lungs.

All intact lipid species with at least one MCSFA and no coeluting species without MCSFA are displayed. Data in heatmap is z-scored and sorted by component 1 of the principal component analysis. Developmental age is shown on the upper x-axis. Each row represents the normalized intensities of a unique chromatographic feature. The features are color coded by row with red indicating high intensity, blue indicating low intensity, and black indicating below limit of detection (LOD, see color key). The underlying numerical data, including lipid species name, used to generate Figure 2 along with statistical significance values is available in Tables S1–7.

Figure 3. LCPUFA abundance in lipid categories and metabolic pathway facilitating the breakdown into eicosanoid products.

(A) The majority of intact lipids with LCPUFA are glycerolipids in the young samples in contrast to the high LCPUFA content in phospholipids in the adult samples. Data in heatmap is z-scored and is sorted by component 1 of the principal component analysis. Developmental age is shown on the upper x-axis. Each row represents the normalized intensities of a unique chromatographic feature. The features are color coded by row with red indicating high intensity, blue indicating low intensity, and black indicating below limit of detection (LOD, see color key). Data shown are all identified lipid species with a fatty acid chain with at least 4 double bonds and at least 20 carbons. Lipid species for which the intensity fell between the LOD for all three replicates of one time point were removed. The underlying numerical data, including lipid species name, used to generate Figure 3a along with statistical significance values is available in Tables S1–8. (B) Metabolic pathway of arachidonic acid metabolism (mmu:00590). The entire metabolic pathway is shown to be increased in adults as compared to PND7 and PND14 mice. Proteins are indicated by diamonds, lipids and lipid metabolites are indicated by circles. Red diamond indicates highest abundances were observed in adult samples, blue diamond indicates highest abundances were observed in PND7 samples, gray diamond indicates that the species was not changing in a statistically significant manner. The lipid metabolites are color-coded by sub-type of eicosanoid metabolite. The entire arachidonic acid metabolic pathway, starting with cleavage from intact phospholipids, is enriched in adults as compared to the younger samples. Expanded view of pathway with protein names and underlying numerical data used to indicate protein expression patterns in the figure as well as statistical significance values is available in Figure S11 and Table S2.

Figure 4. Pro-apoptotic sphingolipid species are observed at highest abundance in mouse lungs undergoing active alveolarization as compared to adult lungs.

L-serine (highest PND7) and palmitoyl-CoA are combined by serine palmitoyltransferase (highest PND7) to begin the de novo pathway of ceramide synthesis. Once ceramide is formed, ceramide species with chain lengths of 14:0, 16:0 and 18:0 are known to induce apoptosis and autophagy (shown in the ceramide heatmap: all pro-apoptotic/autophagic ceramides that change in a statistically significant manner between PND14 and adult). Sphingosine is also a pro-apoptotic molecule that is detected at highest abundance in PND7 mice. This is in contrast to the protein receptors for proliferative sphingosine-1-phosphate (all receptors detected shown in heatmap, S1PR 1–3), which are highest in the adult mice. Sphingolipid-mediated apoptosis is then known to involve the BCL-2/BAX proteins. Shown in the heatmap are the pro-proliferative vs. anti-proliferative BCL-2/BAX family that change in a significantly significant manner between PND14 and adult. All data taken together suggest that sphingolipid-mediated apoptosis occurs in lung tissue undergoing alveolarization. Bar graphs are provided for each lipid, metabolite and protein present in the figure with significance of differential expression across time indicated by stars (see figure legend). The y-axis of each bar graph represent normalized log2 intensity values. The underlying numerical data used to generate Figure 4 along with statistical significance values is available in Tables S1–3 for lipids, Table S2 for proteins and Table S3 for metabolites. In the figure legend, z-score colour coding refers to heatmaps, whereas colour coding for ‘Up PND7’ and ‘Up adults’ refers to arrows and shapes indicating lipids and metabolites.