Metabolic characterisation of THP-1 macrophage polarisation using LC-MS-based metabolite profiling

Abstract

Introduction: Macrophages constitute a heterogeneous population of functionally distinct cells involved in several physiological and pathological processes. They display remarkable plasticity by changing their phenotype and function in response to environmental cues representing a spectrum of different functional phenotypes. The so-called M1 and M2 macrophages are often considered as representative of pro- and anti-inflammatory ends of such spectrum. Metabolomics approach is a powerful tool providing important chemical information about the cellular phenotype of living systems, and the changes in their metabolic pathways in response to various perturbations.

Objectives: This study aimed to characterise M1 and M2 phenotypes in THP-1 macrophages in order to identify characteristic metabolites of each polarisation state.

Methods: Herein, untargeted liquid chromatography (LC)-mass spectrometry (MS)-based metabolite profiling was applied to characterise the metabolic profile of M1-like and M2-like THP-1 macrophages.

Results: The results showed that M1 and M2 macrophages have distinct metabolic profiles. Sphingolipid and pyrimidine metabolism was significantly changed in M1 macrophages whereas arginine, proline, alanine, aspartate and glutamate metabolism was significantly altered in M2 macrophages.

Conclusion: This study represents successful application of LC-MS metabolomics approach to characterise M1 and M2 macrophages providing functional readouts that show unique metabolic signature for each phenotype. These data could contribute to a better understanding of M1 and M2 functional properties and could pave the way for developing new therapeutics targeting different immune diseases.

Keywords: LC–MS; Macrophage polarisation; Metabolic pathway analysis; Metabolite profiling; THP-1 cell.

Fig. 1

Functional categories of the intracellular metabolites produced by THP-1 macrophages. In total, 644 metabolites were putatively identified in THP-1 macrophage extracts using LC–MS-based metabolite profiling

Fig. 2

OPLS-DA scores plots of macrophage extracts after polarisation toward M1 and M2 states and their corresponding unpolarised (M0) controls. a M1 and M2 macrophage samples and their respective unpolarised controls (R² = 0.647 and Q^{2 =} 0.847). b Two-way orthogonal comparison between M0 and M1 (R² = 0.664 and Q² = 0.899). c Two-way orthogonal comparison between M0 and M2 (R² = 0.711 and Q² = 0.838). M0 (yellow circles), M1 (blue squares), and M2 (purple triangles). n = 6

Fig. 3

Summary of pathway analysis by MetaboAnalyst. The top-pathways are ranked by the gamma-adjusted p values for permutation per pathway (y-axis) and the total number of hits per pathway (x-axis). The colour graduated from white to yellow, orange and red as the values of both x and y increase. a Metabolic pathways significantly changed in M1 macrophages. b Metabolic pathways significantly changed in M2 macrophages

Fig. 4

GLs and sphingolipids that significantly affected upon polarisation. a PC (14:2/16:0), b PS (18:0/20:4), and c PE (18:1/22:6) represent some GLs increased significantly in M1 or M2 macrophages compared to M0 macrophages. d Ceramide (d18:1/24:0), e Ceramide (d18:1/22:0) and f Ceramide (d18:1/16:0) as potential key characteristic metabolites of M1 macrophages and their significant increase compared to M0 and M2 macrophages

Fig. 5

Significant metabolic pathways upon polarisation of THP-1 macrophages toward M1 and M2 states with bar graphs of key metabolites that significantly changed, the other metabolites either identified with no significant change (pyruvate, citrate, G6P, glutamate, ornithine and arginine) or unidentified. Metabolic enzymes are as follows; 1: pyruvate dehydrogenase, 2: pyruvate carboxylase, 3: glutamate-aspartate aminotransferase, 4: l-aspartate N-acetyltransferase, 5: N-acetylglutamate synthetase, 6: formiminoglutamase, 7: imidazolonepropionase, 8: tryptophan 5-monooxygenase, 9: arylalkylamine N-acetyltransferase, 10: ornithine aminotransferase, 11: arginase-1, 12: ribosylpyrimidine nucleosidase, 13: pyrimidine-nucleoside phosphorylase. G6P: glucose 6-phosphate, PRPP: 5-phosphoribosyl 1-pyrophosphate, F6P: fructose 6-phosphate. The bar charts show the peak intensity (y-axis) of corresponding metabolites, from the top to the bottom and left to right (cytidine, N-Ac-l-Asp, N-Ac-l-Glu, 4-imidazolone-5-propanoate, 5-hydroxy-tryptophan and N-acetylserotonine). M0 in yellow, M1 in blue and M2 in purple