A metabolic investigation into the effects of various perturbations on human macrophage
Publication Date
July 24, 2020
Creator
Abstract
The “omics” sciences refer to a group of analytical platforms that aim to collectively characterise and quantify the pools of biological molecules such as 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 a biological system. Liquid chromatography (LC)-mass spectrometry (MS)-based metabolomics approach is a powerful tool that has been widely used for disease diagnostics, biomarker discovery and drug toxicity. Moreover, LC-MS-based approaches have 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. In addition, 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 the macrophage spectrum, respectively. 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 compared to naïve macrophages (M0). These data provided functional readouts that show unique metabolic signatures for each phenotype, hence, could contribute to a better understanding of M1 and M2 functional properties, potenially paving 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 profiles of these three subsets of macrophages revealing a distinct metabolic signature for M1 and M2 macrophages compared to M0 macrophages. However, Mreg showed a similar metabolic profile to M0 macrophages. Also, M0 and Mreg showed similar metabolic profiles after challenging them with lipopolysaccharide (LPS) where the significantly changed metabolites were similar to the characteristic key metabolites of M1. Moreover, the plasticity and stability of M1 and M2 macrophages were investigated 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 the possibility of reversing to M1 phenotype showing a similar metabolic profile of M1 macrophages, whereas M1 macrophage did not reverse and their metabolic profiles remain more similar to their original M1 phenotype, indicationg that M1 macrophages are more stable than M2 macrophages.
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 challenged by the secretomes of Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa), and to find the key metabolites in each bacterial infection. Results revealed a distinct metabolic impact of each bacteria. This effect was clear in some metabolic pathways such as glycine and serine metabolism, sphingolipids metabolism, glycerophospholipids (GLs) metabolism, alanine, aspartate and glutamate metabolism and tryptophan metabolism S. aureus and P. aeruginosa showed differential impacts. However, some similarities were shown in the impact on arginine and proline metabolism indicating a different response of macrophages to the secretome of different types of bacteria.
Finally, the ageing process in humans and its consequences on health and society have recently attracted more attention. Therefore, exploring the impact of ageing on macrophages using LC-MS-based global metabolic profiling was conducted on primary macrophages from young and old people in the steady state and in the LPS-stimulated state. However, young and old macrophages showed no difference in their metabolic profiles in both steady state and LPS stimulation, indicating no impact of ageing on macrophages metabolic profile. However, this result could be due to interindividual variabilities or the small sample size.
In conclusion, these results highlight LC-MS-based metabolomics as a promising approach to investigate immune responses under versatile conditions such as polarisation, bacterial infection and ageing. Furthermore, the efficiency of the technique aids the understanding of the mechanisms that underpin these responses, which could be utilised in developing novel therapeutics for manipulating immune diseases.
Item Type
ethesis
Thesis Type
PhD
Associated Schools / Departments
School of Pharmacy (UK)
eprints ID
60312
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