Introduction: Sepsis is a deadly systemic inflammatory response syndrome caused often by pulmonary bacterial infections. Bacteria and its components such as lipopolysaccharides (LPS) recruit large numbers of neutrophils to the airways and can induce the release of cytotoxic neutrophil extracellular traps (NETs) from neutrophils. Neutrophils and NETs are major regulators of sepsis. Our lab has previously shown that Actinomycin D (Act D), a DNA intercalating compound extensively used in clinics, can suppress NET formation induced by various agonists (e.g., LPS).

Methods and Results: Here we show that pulmonary pathogens such as Staphylococcus aureus (Gram-positive bacteria) and Pseudomonas aeruginosa (Gram-negative bacteria) induced NETosis with increasing multiplicity of infection (MOI; 0-50) of the bacteria, as determined by Sytox Green assays and immunoconfocal microscopy (myeloperoxidase, citrullinated histone H3); Act D suppressed the NETosis induced by both of these bacteria at different MOIs. We tested whether Act D have any direct effect on bacterial killing or phagocytic killing. Act D neither directly affected the survival of bacteria nor the phagocytosis and microbial killing by neutrophils (colony-counting assays). In vivo experiments showed that Act D reduced NET formation in the airways of LPS-instilled mice (bronchoalveolar lavage; Western blots).

Conclusions: These studies show that Act D suppresses NETosis without affecting the anti-microbicidal functions of neutrophils, and hence, represents a potentially life-saving therapeutic compound for treating sepsis.