Abstract
Immunometabolism is an emerging field of research that aims to understand how the metabolic state of immune cells influences their fate and function, highlighting the interconnection between metabolism and immune responses in health and disease. During activation and differentiation, immune cells undergo metabolic reprogramming. This involves shifting between key metabolic pathways, including glycolysis and mitochondrial respiration, to meet their functional demands. Dysregulation of these pathways has been linked to chronic inflammatory autoimmune diseases, including rheumatoid arthritis (RA).
Research into immunometabolism has shown that people with RA have distinct metabolic profiles in immune cells, particularly T cells, that regulate their activation, differentiation and effector function. Methotrexate (MTX) is the gold-standard treatment for RA, although up to 40% of patients respond inadequately within the first two years. The MTX mechanism of action involves both immune and metabolic modulation. However, in the context of RA and MTX, the specific anti-inflammatory and immunometabolic pathways involved are unclear.
This honours research project aimed to optimise and establish an immunometabolism protocol, SCENITH (Single-Cell ENergetic metabolism by profIling Translation inHibition), for use in human whole blood. The goal of using this method was to characterise metabolic pathways of healthy human participants’ immune cells, both untreated and treated ex vivo, with MTX.
SCENITH was successfully optimised for human whole blood by determining the optimal concentrations and durations for metabolic inhibitors and treatments, as well as the appropriate antibody titre, while minimising background signal on a spectral flow cytometer. Application of the optimised SCENITH protocol in healthy human whole blood revealed distinct metabolic signatures between the analysed immune cell subsets, which aligned with their functional roles. Interestingly, ex vivo MTX treatment altered the metabolic profiles of specific blood immune cells, particularly T cell subsets and CD56bright NK cells, indicating a change in metabolic dependencies and capacities compared to untreated immune cells. Therefore, modulation of immune cell metabolism may be a key component of MTX’s anti-inflammatory effects.
The optimisation and application of SCENITH in healthy human whole blood sheds light on immune cell metabolic profiles and presents preliminary evidence that MTX affects immunometabolism. These findings provide a foundation for further studies to compare metabolic signatures between healthy individuals and people living with RA, as well as the effect of MTX treatment.