Synthetic Oligodeoxynucleotides in the Regulation of Neutrophil Apoptosis

Abstract

Synthetic oligodeoxynucleotides (ODNs) mimic the immunostimulatory activity of viral/microbial/host DNA. The present study was undertaken to investigate the effects of ODNs on polymorphonuclear leukocyte (PMNL, neutrophil) apoptosis. Neutrophils are essential for innate immunity, and the ability of ODNs to influence neutrophil functions makes them promising for use in immunotherapy. Little is known about the effects of different ODN structures on neutrophil life and death. We studied how ODNs affect both unconstrained and bacterially suppressed apoptosis of neutrophilic granulocytes. Apoptosis modulation by phosphodiester ODNs and by structures with different numbers of phosphorothioate substitutions was compared. In the case of CpG-ODNs, it was the compounds with phosphorothioate bonds that had a pronounced proapoptotic effect upon short-term (4-6 hours) exposure. After 24 hours, the most effective were A-class CpG-ODNs, with phosphodiester inner core and phosphorothioate oligoguanosines (poly(g)) attached to the ends, as well as telomeric phosphodiester (TTAGGG)n repeats flanking with 3’- and 5’- poly(g) sequences, like those in A-class CpG-ODNs. Intracellular oxidant accumulation often triggers apoptotic pathways in neutrophils. Phosphorothioate-containing CpG-ODNs concomitantly stimulated production of nitric oxide and superoxide, which may rapidly combine to generate peroxynitrite. ODNs with phosphodiester bonds did not activate oxidant production. We experimentally showed the anti-apoptotic activity of bacteria Salmonella typhimurium. CpG-ODNs prevented bacteria-associated inhibition of apoptosis at doses that do not affect the lifespan of resting cells. The pro-oxidant effect of low concentrations of CpG ODNs was not sufficient to trigger irreversible pro-apoptotic mechanisms, but the sensitivity of PMNLs to ODNs as modulators of apoptosis, increased significantly under conditions of infectious inflammation. Telomeric phosphodiester (TTAGGG)4 repeats with phosphorothioate oligoguanosines attached to the ends were less effective in prevention of bacteria-associated inhibition of apoptosis than A-class CpG-ODNs with the same modified oligoguanosines overhangs. The results of our study revealed that synthetic analogs of bacterial/host DNA, when phosphorothioate modified, can be used as inducers of reactive oxygen species generation by neutrophils. This allows to overcome the excess pro-survival effect caused by some pathogens, in particular Salmonella typhimurium, thus facilitating the successful resolution of inflammation. Ethics statement. The authors prepared neutrophils from the blood of healthy volunteers. Blood was collected via venous puncture, as approved by the Ministry of Public Health Service of the Russian Federation. Experimental and the subject consent procedures were approved by the Ethics Committee of the A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University. Fully informed consent was obtained, and all investigations were conducted according to the principles of the Declaration of Helsinki.