Background: Plasmacytoid dendritic cells (pDCs) play pivotal role in antiviral immunity, as highly potent producers of type I interferons (IFNs). Their early viral detection and type I IFN production are mediated by endosomal Toll-like receptors (TLRs), whereas a second wave of type I IFN responses is guided by cytosolic retinoic acid-inducible gene-I (RIG-I), the expression of which is up-regulated in pDCs upon TLR stimulation. Generation of mitochondrial reactive oxygen species (mtROS) triggered by stress or metabolic changes in the cells is one of the key regulators of virus-stimulated signaling pathways. In this work, we studied the effects of mtROS on TLR9-induced and especially on RIG-I-mediated antiviral responses of pDCs.
Methods: TLR9 or RIG-I ligand-activated pDCs were treated with Antimycin A, a specific inhibitor of mitochondrial electron transport chain at complex III increasing the level of mtROS in the cells, or exposed to H2O2. The mtROS production was assessed using a fluorogenic superoxide indicator targeted to mitochondria, while cellular viability was determined by 7-AAD staining. Changes in antiviral signaling pathways were detected by Q-PCR and western blot.
Results: Elevated level of mtROS markedly reduced the TLR9 agonist-induced expression of type I IFN genes in pDCs, similarly to the effect of exogenous H2O2 treatment. Interestingly, TLR9-induced expression of RIG-I was negatively regulated by mtROS, but not by exogenous H2O2. In contrast, mtROS further enhanced the expression of type I IFN genes induced by RIG-I agonist in pDCs as well as the expression of MAVS and the phosphorylation of Akt, the essential components in the RIG-I signaling pathway.
Conclusion: Collectively, our data indicate that mtROS have notable and different modulatory effects on TLR- and RIG-I-mediated IFN production providing a novel potential therapeutic tool for regulation of antiviral responses in pDCs both at early and late phases of infections.

This work was supported by the Hungarian Scientific Research Fund (OTKA PD 115776 to K.P.) and K.P. was also supported by the János Bolyai Research Scholarship from the Hungarian Academy of Sciences.