Introduction: Plasmacytoid dendritic cells (pDC) play a crucial role in innate viral immunity as the most potent producers of type I interferons (IFN-I) in the human body. However, the metabolic regulation of IFN-I production in such vast quantity remains poorly understood, with conflicting evidence in the literature.
Methods: To investigate the role of metabolic pathways in IFN-α production, we employed flow cytometric methods and extracellular flux analysis (EFA) to measure changes in metabolic activity downstream of TLR7 and -9 ligation. We also assessed the effect of metabolic inhibitors on IFN-α expression.
Results: Basal oxygen consumption and respiratory capacity both significantly increased upon 6 h HSV or flu stimulation, indicating an increase in the rate of and capacity for aerobic metabolism. Smaller-scale, statistically significant changes in extracellular acidification were also observed. Likewise, mitochondrial membrane potential significantly increased under the same conditions, while small but significant increases were also apparent in 2-NBDG uptake, a fluorescent glucose analog taken up through glucose transporters with similar kinetics.
IFN-α production correlated with these changes in metabolic activity: we showed significant IFN-α inhibition with separate treatments of the inhibitors etomoxir (carnitine palmitoyltransferase 1), UK5099 (mitochondrial pyruvate translocase), and 2-deoxyglucose (hexokinase). In addition, IFN-α production was completely ablated 6 h post-flu stimulation with treatment with the inhibitors oligomycin (ATP synthase), rotenone (complex I), and antimycin A (complex III). Cell viability was unaffected by all treatments, suggesting a necessity of mitochondrial oxidation in the production of IFN-α.
Conclusion: We have demonstrated evidence for the necessity of mitochondrial oxidation in the innate antiviral response of pDC, the inference being that the increase in ATP production provides energy for the translation of large amounts of IFN. Our data also suggest a role for glycolysis, which likely relates to the resultant pyruvate as a major substrate of the TCA cycle.