Phosphorothiorate Oligodeoxynucleotides Induce Antimicrobial Epithelial Mitochondrial Reactive Oxygen Species That Protect Against Pneumonia

Background: Pneumonias exert tremendous worldwide morbidity, especially among immunocompromised populations. Our laboratory has identified an inhaled therapeutic comprised of Toll-like receptor (TLR) 2/6 and TLR9 agonists that protects mice against a broad array of respiratory pathogens. This protection is associated with the generation of an antimicrobial environment in the lungs and requires induction of epithelial reactive oxygen species of both dual oxidase and mitochondrial sources. Objective: To discern how the treatment induces generation of mitochondrial reactive oxygen species (mtROS). Results: Inducible mtROS generation was driven by epithelial exposure to a class C CpG oligodeoxynucleotide (ODN M362), a reported TLR9 ligand. However, ODN M362-induced mtROS generation was not dependent on TLR9 signaling, but required that the oligodeoxynucleotide include phosphorothiorate linkages, rather than the phosphodiester linkages common in the DNA of most species. Phosphorothiorate ODN directly interacted with mitochondrial permeability transition pores, altering the ATP:ADP ratio in both cytosol and mitochondria, activating AMPK and ACC pathways and increasing beta-oxidation. This led to enhancement of electron transport chain complex II activity and reduction of complex III activity, causing increased electron leakage that resulted in superoxide (O2- ·) production. The resultant mtROS both exerted directly microbicidal effects and stabilized Hif1α to support antimicrobial peptide production. Conclusions: These data identify novel means to manipulate mtROS and may provide an opportunity to protect vulnerable patients against pneumonia.