Lipidomic characterization and localization of phospholipids in the human lung

Abstract

Lipids play a central role in lung physiology and pathology; however, a comprehensive lipidomic characterization of human pulmonary cells relevant to disease has not been performed. The cells involved in lung host defense, including alveolar macrophages (AMs), bronchial epithelial cells (BECs), and alveolar type II cells (ATIIs), were isolated from human subjects and lipidomic analysis by LC-MS and LC-MS/MS was performed. Additionally, pieces of lung tissue from the same donors were analyzed by MALDI imaging MS in order to determine lipid localization in the tissue. The unique distribution of phospholipids in ATIIs, BECs, and AMs from human subjects was accomplished by subjecting the large number of identified phospholipid molecular species to univariant statistical analysis. Specific MALDI images were generated based on the univariant statistical analysis data to reveal the location of specific cell types within the human lung slice. While the complex composition and function of the lipidome in various disease states is currently poorly understood, this method could be useful for the characterization of lipid alterations in pulmonary disease and may aid in a better understanding of disease pathogenesis.

Keywords: alveolar macrophages; alveolar type II cells; bronchial epithelial cells.

Fig. 1.

Lipidomic LC/MS and LC/MS/MS analysis to identify phospholipid molecular species, including fatty acyl substituents in human pulmonary cells. A: Extraction of m/z 885.7, PI(18:0_20:4), from the negative ion NP-HPLC/MS analysis of the Bligh-Dyer extract of human BECs. B: CID of m/z 885.7 from the negative ion NP-HPLC/MS/MS analysis of the Bligh-Dyer extract of human BECs.

Fig. 2.

Volcano plots of differentially abundant lipids in human pulmonary cells identified by mass spectrometric lipid analysis. The −log10 t-test P values were plotted against the fold change in mole fraction abundances of the phospholipid molecular species. The horizontal green line represents a statistical significance of 0.05 and the pink vertical lines represent the phospholipid molecular species up or down 2-fold in the two compared cell types. The data points in purple have a P value >0.05 and, therefore, are not significantly different in the two types of cells compared, while those data points in orange are significantly different (P < 0.05), but have a small fold change (<2-fold) between the two types of cells. When comparing two types of cells, the blue data points indicate phospholipids that are significantly abundant by more than 2-fold in the first cell type and the red data points reveal phospholipid molecular species that are significantly abundant by more than 2-fold in the second cell type. BECs versus ATIIs (A), AMs versus ATIIs (B), and AMs versus BECs (C).

Fig. 3.

Mole fraction abundances of specific phospholipid molecular species in ATIIs, AMs, and BECs of interest determined from the volcano plots in Fig. 2 and MALDI images of the same molecular species in human lung slices. The MALDI images for PG, PI, and PE molecular species are all from the same lung slice in negative ion mode and the MALDI images for the PC molecular species are in positive ion mode from an adjacent lung slice. PG(16:0_18:1) (A), PC(16:0_16:0) (B), PE(O-16:1_20:4) (C), PC(O-16:0_20:4) (D), PC(18:0_20:4) (E), and PI(18:0_20:4) (F).

Fig. 4.

Comparative localization of phospholipid molecular species in a human lung slice. A: Modified Giemsa stain of the human lung slice after MALDI IMS in negative ion mode. B: Merged negative ion MALDI image of PE(O-16:1_20:4) (green) at m/z 722.6, PI(18:0_20:4) (red) at m/z 885.6, and PG(16:0_18:1) (blue) at m/z 747.6.