Figure 5

Butyrate impacts NK cell metabolic machinery. Purified NK cells were cultured for 18 h in the absence or presence of IL-12 (30 ng/mL) and IL-15 (100 ng/mL). Butyrate (1 mM) was added where indicated for the duration of the experiment. Measurement of cellular bioenergetics (respiratory activity) was performed using the Seahorse extracellular flux analyzer. (a) Oxygen consumption rate (OCR) and (b) extracellular acidification rate (ECAR), data displayed as boxplots with bar representing the mean and error bars the SEM (n = 5). PBMCs were cultured for 18 h in the absence or presence of IL-12 (30 ng/mL) and IL-15 (100 ng/mL). Butyrate (1 mM) was added where indicated for the duration of the experiment. CD71 expression by was determined by flow cytometry. (c) Representative dot plots and (d) individual paired data of CD71 expression by CD56^bright and CD56^dim NK cells, is shown (n = 6). (e) Mitochondrial mass of NK cells was measured by MitoTracker Green staining of PBMCs and gating on CD56^bright and CD56^dim cells. (f) Mitochondrial membrane potential analysis was performed by staining PBMC with TMRM (100 nM) for 30 min at 37 °C and gating on CD56^bright and CD56^dim NK cells. Individual datapoints are shown and bars represent mean and error bars, SEM (n = 5–7). (g) Purified NK cells were cultured with IL-12 (30 ng/mL) and IL-15 (100 ng/mL) ± Butyrate (1 mM) as described above. Expression of hexokinase 2 (HK2) was assessed by qRT-PCR (n = 7–10). Individual datapoints are shown, bars represent the average value ± SEM. Samples were compared using either a one-way ANOVA followed by a Kruskal–Wallis post hoc test or Wilcoxon’s signed rank test, as appropriate *p < 0.05, **p < 0.01, ***p < 0.001.