Quantitative profiling of glycerophospholipids during mouse and human macrophage differentiation using targeted mass spectrometry

Abstract

Macrophage lipid metabolism plays a pivotal role in innate and adaptive immune responses. Previous studies have shown that this process plays a role in infections and contributes to the pathogenesis of diabetes, atherosclerosis, and other immunometabolic diseases. M1 macrophages, or classically activated macrophages, are key players in the defense against bacterial infections. M2 macrophages, or alternatively activated macrophages, are involved in anti-inflammatory responses. Using the multiple reaction monitoring method, we identified changes in lipid composition during the differentiation of human and murine macrophages. We detected over 300 lipid molecules in mammalian macrophages, and we observed a striking shift in the composition of glycerophospholipids (GLs) from saturated and monounsaturated to polyunsaturated during human macrophage polarization. Moreover, M2 macrophages showed a higher level of lysophospholipids (lysoGLs) than did M1 macrophages. The lysoPI species increased in human and mouse M2 macrophages, suggesting that they may be involved in M2 macrophage polarization and anti-inflammatory processes. Collectively, these results indicate that lipids may play a role in the pro- and anti-inflammatory activities of macrophages and may be markers of the macrophage activation state.

Figure 1

Schematic diagram of macrophage polarization and lipid detection.

Figure 2

Activated macrophages derived from human monocytic THP-1 cells show an increase in lysoPE, PE, and PG and a decrease in lysoPG. (a) Left to right, cd11b and cd36 were analyzed to identify the differentiation of inactivated macrophages. The expression levels of ccl3, tnf-a, ccl22, and ccl17 were analyzed to assess the polarization into M1 and M2 macrophages. The data represent the mean ± SEM, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001. (b,c) Lipids were extracted from macrophages in different differentiation stages and then identified using the MRM method. The lipid classification between two groups was compared using Student’st-test. Histogram and clustering analysis demonstrated increased lysoPC and lysoPS in inactivated macrophages compared to THP-1 monocytes. Activated macrophages, including M1 and M2 cells, showed increased levels of PE, lysoPE, and PG and downregulation of lysoPG. A comparison of M1 and M2 cells indicated that the lipid classification of lysoPI was dramatically increased in M2, whereas that of lysoPS was increased in M1 cells. (d) PCA indicated that four groups of cells could be separated from each other in a 2D score plot (up) and further divided in a 3D score plot (below).

Figure 3

Activated macrophages derived from human monocytic THP-1 cells show the accumulation of medium- and long-chain polyunsaturated lipids (a,b) Histogram (a) and clustering analysis (b) indicated that during macrophage differentiation, long-chain and polyunsaturated lipids (PS38:3, PS38:5, PS40:6, PC40:6, and PI38:4, etc.) accumulated in the activated macrophages. In contrast, saturated and monounsaturated lipids with fewer carbons (PS32:1, PC32:1, PI30:0, PI34:1, and PI36:1, etc.) were elevated in THP-1 and M0 cells (* represents comparisons between M1 and M0, # represents comparisons between M2 and M0). (c) Volcano plots show that lipids were differentially expressed between M1 and M2 macrophages. The x-axis shows the log2-fold change (fold change = intensity of M2 to M1). The y-axis shows the -log10 p-value for the corresponding lipids. The top lipids that are elevated in M1 cells are highlighted in green, and those that are elevated in M2 cells are indicated by red dots (fold change >1.5 or < 0.67, p < 0.05).

Figure 4

Most glycerophospholipids and lysophospholipids are increased in mouse activated macrophages. (a) From left to right, the expression levels of nos2, cxcl10, arg-1, and mrc-1were analyzed to assess the polarization into M1 and M2 macrophages. The data represent the mean ± SEM, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001. (b,c) Histogram and clustering analysis show a comparison of the lipid classification between the two groups. Most glycerophospholipids and lysophospholipids were elevated in activated macrophages. Ceramide and ceramide-1-phosphate were also increased in activated cells. (d) PCA suggested that three types of macrophages could be separated from each other in 2D and 3D score plots.

Figure 5

Mouse M2 macrophages have higher levels of lysophospholipids than do M1 macrophages. (a,b) As shown in the histogram and heatmap, lysophospholipids, including lysoPI, lysoPG, and lysoPS, were increased in M2 macrophages, whereas PI, PG, and PS were increased in M1 macrophages. (c) Volcano plots show that lipids were differentially expressed between M1 and M2 macrophages. The x-axis shows the log2-fold change (fold change = intensity of M1 to M2). The y-axis shows the-log10 p-value for the corresponding lipids. The top lipids that are elevated in M1 cells are highlighted in red, and those that are elevated in M2 cells are indicated by green dots (fold change >1.5 or <0.67, p < 0.05).