Abstract Body: Background: Despite advancements in research and public health initiatives over the last 50 years, hypertension (HTN) remains a burden in the US. Chronic activation of T cells in HTN skews their development toward an inflammatory phenotype (i.e. Th1, Th17) and away from a more protective path (Treg). Further, T cells have been observed in various end organs of hypertensive mice and humans where they release mediators of inflammation and cause fibrosis. Over half of immune cell energy is allocated to protein synthesis (PS). Therefore, we sought to identify a T cell subset whose metabolic profile catalyzes the promotion of inflammation in HTN using Single Cell ENergetic metabolism by profilIng Translation InHibition (SCENITH).
Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from human whole blood samples and cryopreserved. Thawed PBMCs were stimulated overnight, plated, and treated in tandem with metabolic inhibitors 2-deoxy-D-Glucose (DG, inhibits glycolysis) and oligomycin A (O, inhibits OXPHOS) to form the following groups: + Control (no inhibitors), DG, O, and DG+O. Samples were treated with puromycin (puro), an antibiotic that resembles a tRNA and is incorporated into cell translation yielding truncated, puro-positive polypeptides. Cells were stained and analyzed using spectral flow cytometry. Inclusion of anti-puro antibody allowed for measurement of puromycin mean fluorescence intensity (PuroMFI) which served as a direct readout for PS. PuroMFI of each group was used to calculate glucose dependence (GD), mitochondrial dependence (MD), glycolytic capacity (GC), and fatty acid oxidation/amino acid oxidation capacity (FAO/AAO).
Results: Using a 29-color human metabolic panel, we profiled 18 distinct immune cell populations in just one SCENITH experiment. Amongst these, we witnessed striking metabolic differences between normotensive and hypertensive Th17s. Hypertensive Th17s have reduced GD compared to normotensives (p<0.05). In addition, hypertensive Th17s have increased FAO/AAO capacity compared to normotensives (p<0.05).
Conclusions: We have preliminarily revealed decreased GD and increased FAO/AAO capacity in hypertensive Th17s compared to normotensives. Less dependence on glucose allows for ATP production and synthesis of pro-inflammatory cytokines (i.e. IL17A) using FA/AAs when other methods of metabolism are unavailable. Thus, we have demonstrated the novel use of SCENITH for single-cell metabolic profiling of immune cells in HTN.