The Role of ecSOD in Neutrophil Containment of Listeria Monocytogenes

Background: Extracellular superoxide dismutase (ecSOD) is an antioxidant that serves to minimize host tissue damage during reactive oxygen species (ROS) mediated immune responses. Listeria monocytogenes (LM) is an intracellular bacteria that is often used to study host pathogen interactions during intracellular bacterial infections. Previously, we showed that ecSOD activity is detrimental to the host during infection with LM. Furthermore, using depletion studies, we determined that neutrophils, a set of innate immune cells which are known for ROS generation, from mice that lack ecSOD (ecSOD KO) are more protective during LM infection. Materials and Methods: Using ecSOD congenic mice (expressing differing activities of ecSOD as indicated by their titles; ecSOD HI, ecSOD Wild Type, ecSOD Knockout), flow cytometry and a unique LM termed actA:LMGFP, we set out to determine how ecSOD activity modulates the protective capabilities of neutrophils during LM infection. Hypothesis: We hypothesized that ecSOD activity would hinder the ability of neutrophils to keep LM contained in the phagosome and therefore suppress their ability to ultimately kill the bacteria. Results: In vitro, a higher percentage of neutrophils from the liver, spleen, bone marrow, and peritoneal cavity ecSOD KO mice, allowed for phagosomal escape in comparison to the ecSOD expressing neutrophils. In vivo, at a high dose of infection, a similar trend was observed in the spleen, although, the opposite occurred in the liver. However, using MFI as an indicator of the relative number of bacteria per neutrophil, we observed that the ratio of cell associated to cytosolic bacteria was higher in ecSOD KO neutrophils in comparison to neutrophils with ecSOD activity. The next step was to determine if ecSOD modulates neutrophil protective mechanism downstream of phagosomal containment, mainly, autophagy. It was observed that phagosomal escape correlates with the initiation of autophagy in bone marrow neutrophils. However the effect on neutrophils from other organs is currently inconclusive. Conclusions: EcSOD activity does appear to modulate neutrophil association with LM. Lack of ecSOD activity increases association of the bacteria with neutrophils but simultaneously decreases the phagosomal escape of the bacteria into the cytosol. However, the presence of cytosolic bacteria induces the initiation of autophagy as a mechanistic means of keeping LM contained which should eventually lead to killing by the neutrophil.