In-depth immunometabolic profiling by measuring single-cell protein translation inhibition via bioorthogonal noncanonical amino acid tagging (SCENITH-BONCAT)

Abstract

AbstractMotivationSeahorse Extracellular Flux (XF) analysis has been influential in immunometabolism research, measuring cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) to determine immune cell metabolic profiles. However, Seahorse XF has several limitations, relying on purified adherent cells, relatively high cell numbers, and specialized equipment and reagents. Recently, a novel flow cytometry-based technique called SCENITH (Single Cell Energetic metabolism by profiling Translation inhibition) was introduced, which measures cellular protein synthesis through puromycin staining as a proxy for metabolic activity in single cells. The current limitation of SCENITH is its reliance on a specific anti-puromycin antibody that is not commercially available. To address this, we propose an alternative approach using biorthogonal noncanonical amino acid tagging (BONCAT) to measure protein synthesis.SummaryThe field of immunometabolism has revealed that cellular energy metabolism significantly contributes to immune cell function. Disturbances in immune cell metabolism have been associated with various diseases, including obesity, atherosclerosis, and cancer. To further advance immunometabolic research, developing novel methods to study the metabolism of immune cells in complex samples is required. Here, we introduce SCENITH-BONCAT, an adapted version of SCENITH which utilizes click-labeling of homopropargylglycine (HPG), an alkyne-bearing methionine analog, to measure protein synthesis as a proxy of metabolic activity. SCENITH-BONCAT successfully reproduced known metabolic signatures of immune cell activation. Specifically, LPS/IFNγ-induced classical activation increased glycolytic capacity, and IL-4-induced alternative activation enhanced mitochondrial dependence in human macrophages. The assay’s applicability was further explored in peripheral blood mononuclear cells (PBMCs) from healthy volunteers, revealing diverse metabolic rewiring in immune cell subsets upon stimulation with different activators. Cryopreserved PBMCs showed reduced HPG incorporation and mitochondrial dependence compared to freshly isolated cells, warranting attention to cellular mitochondrial health during cryopreservation. Finally, SCENITH-BONCAT was used to analyze murine tissue-resident immune cells from different organs. Principal component analysis (PCA) revealed tissue-specific clustering based on metabolic profiles, likely driven by microenvironmental priming of tissue-resident immune cells. In conclusion, SCENITH-BONCAT offers valuable insights into immune cell metabolic responses and presents a powerful platform for studying immune cell metabolism in complex samples and tissue-resident immune populations in both human and murine studies.