Immunology
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21687
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Browsing Immunology by Author "Berg, Rance E."
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Item The effect of ecSOD on HMGB1 and it's role during Listeria infection(2017-03-14) Witter, Alexandra; Okunnu, Olubusola; Berg, Rance E.; Swanta, NaomiBackground: Listeria monocytogenes (LM) is a gram-positive, intracellular foodborne pathogen which can cause severe disease in immunocompromised individuals and is a leading cause of death from foodborne infection. During LM infection, reactive oxygen species (ROS) represent a first line of defense, which not only destroys the pathogen but can also cause collateral tissue damage. Extracellular superoxide dismutase (ecSOD) is a potent antioxidant that protects host tissue by regulating ROS concentrations. Using congenic mice that express varying levels of ecSOD, our lab has previously demonstrated that ecSOD activity decreases resistance to LM infection and pro-inflammatory cytokine production. In contrast, ecSOD enhances neutrophil recruitment to the liver during LM infection. High-mobility group box 1 protein (HMGB1) is a ROS modulated protein, which has been shown to promote neutrophil recruitment when critical cysteines are reduced but promotes pro-inflammatory responses when these residues are oxidized. We hypothesize that ecSOD activity reduces cysteine residues in HMGB1, thus enhancing neutrophil recruitment while decreasing pro-inflammatory responses during LM infection. Purpose: The purpose of our study is to understand the effect ecSOD has on transcription, translation and post-translational modification of HMGB1. We are also studying the effect of HMGB1 on inflammatory cytokine production such as TNFa, IL-1 and IL-6 and neutrophil recruitment to infected organs during LM infection in ecSOD congenic mice. Methods: C57BL/6 mice were infected with LM and at 3 days post infection immune cells were isolated from the spleen and bone marrow. The cells were used for rt-PCR to study HMGB1 transcription, and western blotting to study the translation of HMGB1. Furthermore, the aforementioned cells from ecSOD congenic mice were also incubated overnight with 10ng/mL of HMGB1 and supernatants were harvested to measure TNFa by ELISA. Results: Our results indicate that LM does not have a significant effect on HMGB1 transcription and translation in the spleen and bone marrow. We also show that at 10ng/mL, HMGB1 does not enhance or induce TNFa production in spleen and bone marrow cells from ecSOD congenic mice. Conclusions: Our results indicate that HMGB1 production is not influenced by LM infection in C57BL/6 mice. Furthermore, our data suggest that HMGB1 may not influence immune responses against LM in ecSOD congenic mice in vitro. Further studies are required to elucidate the in vivo functions of HMGB1 during LM infection.Item The Role of ecSOD in Neutrophil Containment of Listeria Monocytogenes(2017-03-14) Swanta, Naomi; Berg, Rance E.; Okunnu, BusolaBackground: 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.