Dynamic metabolic reprogramming in dendritic cells: An early response to influenza infection that is essential for effector function
Fig 5
Limiting glycolysis or mitochondrial oxidation of pyruvate, glutamine or long chain fatty acids impairs infected DC function.
(A) DC were seeded on precoated Oris 96-well motility plate and allowed to adhere for 18+ hours followed by 4 hours of inhibitor treatment (2 μM c-Myc inhibitor, 3 μM BPTES, 4 μM etomoxir or 2 μM UK5099), plug removal and infection (MOI = 5) for 17 hours. Cells were stained with Calcein-AM (2 μM), and viability and motility were determined with fluorescence at 485/528 nm. The graph represents the mean values of 3 experiments +/- SD. (B-C) CD8+ T cells were isolated by negative depletion from fresh splenocytes of female Ot1 mice and co-cultured with DC at 5:1 ratio T cells to DC for 24 hours. Basal ECAR was established, and T cell activation by anti-CD3/CD28-coated DynaBeads was monitored for 2 hours and maximal ECAR determined. (B) One representative graph is shown from 3 independent experiments. (C) The graph represents the mean values of 3 experiments +/- SD. (D-F) CD8+ T cells were isolated by negative depletion from fresh splenocytes derived from C57BL/6-Tg(TcraTcrb)1100Mjb/J (OT1) and co-cultured with DC at 5:1 ratio of T cells to DC for 24 hours. T cell were then stained for CD8, CD45.1, CD25, and CD44 and enumerated with flow cytometry. We selected CD8 and CD45.1 positive (D) and then gated on CD25 (E) and CD44 (F). The graph represent the mean values of 3 experiments +/- SD. Significant differences among means were found with ANOVA followed by Tukey's multiple comparisons test with asterisks indicating adjusted p-values (* p≤0.05, ** p≤0.001, *** p≤0.0001, and **** p<0.0001).