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The “omics" sciences refer to a group of analytical platforms that aim to collectively characterise and quantify the pools of biological molecules, genes (genomics), transcripts (transcriptomics), proteins (proteomics) and metabolites (metabolomics), which will be translated into the structure and the function of organisms, tissues or cells. Metabolomics concerns with identification and quantification of small molecules that represent the end products of the biological processes in the biological samples. Liquid chromatography

(LC)-mass spectrometry (MS)-based metabolomics approach is a powerful tool that has been widely used for disease diagnostic, biomarker discovery and drug toxicity. Moreover 

LC-MS-based approach has been employed to investigate the function of immune cells and immune responses by monitoring level changes of signalling molecules. Macrophages constitute a heterogeneous population of functionally distinct immune cells involved in several physiological and pathological processes. They play a crucial role in innate and adaptive immunity to protect the body from any invading danger. Macrophages display remarkable plasticity by changing their phenotype and function in response to environmental cues displaying a wide spectrum of different functional phenotypes.

In this thesis, untargeted LC-MS-based metabolite profiling was applied to characterise 

THP-1 macrophages and differentiate between M1 and M2 phenotypes to identify key characteristic metabolites for each polarisation state. The so-called M1 and M2 macrophages are often considered as the extremes of polarisation states and representative of pro- and anti-inflammatory ends of macrophages spectrum. The results showed that M1 and M2 macrophages have distinct metabolic profiles. Sphingolipid, pyrimidine and purine metabolism were significantly changed in M1 macrophages, whereas arginine, proline, alanine, aspartate and glutamate metabolism were significantly altered in M2 macrophages. These data provided functional readouts that show unique metabolic signature for each phenotype, hence, 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.

In the second part of this thesis, the investigation of THP-1 macrophages polarisation and plasticity were extended to encompass the functional classification of macrophages; classically activated macrophages (M1), wound healing macrophages (M2) and regulatory macrophages (Mreg). LC-MS-based global metabolic profiling was carried out to differentiate between the metabolic signature of these three subsets of macrophages revealing a distinct metabolic signature for M1 and M2 macrophages compared to naive macrophages. 

However, Mreg showed similar metabolic profile to M0 macrophages. Also, M0 and Mreg showed similar metabolic profile upon treating with lipopolysaccharide (LPS) of which the significantly changed metabolites similar to the characteristic key metabolites of M1. Moreover, the plasticity and stability of M1 and M2 macrophages were investigates using

LC-MS-based global metabolic profiling after treating M1 macrophages with M2 inducer cytokines and vice versa. The results revealed that M1 macrophages are more stable than M2 macrophages. However, M2 showed possibility of reversing to M1 phenotype showing similar metabolic profile of M1 macrophages, whereas M1 macrophage didn't reversed and their metabolic profile remain more similar to their original M1 phenotype.

In the third part of this thesis, LC-MS-based global metabolic profiling was used to distinguish between the impact of Gram-positive bacteria and Gram-negative bacteria on THP-1 macrophages represented by Staphylococcus aureus (S.aureus) and Pseudomonas aeruginosa (P.aeruginosa), respectively, and find the key metabolites in each bacterial infection. Results revealed distinct metabolic impact of each bacteria. This effect was clear in some metabolic pathways such as Glycine and serine metabolism, sphingolipids metabolism, GLs metabolism, alanine, aspartate and glutamate metabolism and tryptophan metabolism S.aureus and P.aeruginosa showed differential impact. However, some similarities were shown in the impact on arginine and proline metabolism.

Finally, aging process in human and its consequences on the health and society have recently attracted more attention. Therefore, exploring the impact of aging on macrophages using 

LC-MS-based global metabolic profiling was conducted on young's and elderlies' macrophages in the steady state and in the LPS-stimulated state. Young's and elderlies' macrophages showed no difference in their metabolic profile in both steady state and LPS stimulation.

In conclusion, these results highlight the LC-MS-based metabolomics as a promising approach to investigate immune response under versatile conditions such as polarisation, bacterial infection and aging. Furthermore, its efficiency in understanding the mechanisms that underpin these responses, which could be utilised in developing novel therapeutics