Browsing by Author "Break, Timothy J."
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Item Divergent Roles of Extracellular Superoxide Dismutase During Intracellular Bacterial Infection(2016-03-23) Break, Timothy J.; Okunnu, Busola; Swanta, Naomi; Berg, Rance E.; Witter, AlexandraExtracellular superoxide dismutase (ecSOD) regulates extracellular concentrations of reactive oxygen species (ROS) to protect tissues during infection and inflammation. Using congenic mice with varying levels of ecSOD activity (ecSOD HI, WT and KO), we have previously shown that ecSOD activity enhances neutrophil recruitment to the liver, yet inhibits the innate immune response against Listeria monocytogenes (LM) leading to increased host susceptibility. Additionally, we determined that ecSOD activity protects the extracellular matrix (ECM) from degradation and promotes egress of immature neutrophils out of the bone marrow and into the liver where they are unable to provide protection against LM. Since ecSOD can be produced by cells from the hematopoietic lineage as well as somatic cells, the potential contribution of ecSOD produced by cells from each lineage required further investigation. In order to determine the relative contributions of ecSOD produced from either hematopoietic-derived cells or somatic cells, we generated bone marrow chimera mice using ecSOD KO mice and C57Bl/6 mice. Briefly, host mice were irradiated to eliminate hematopoietic lineage cells and reconstituted with bone marrow cells isolated from donor mice. Control groups consisted of ecSOD KO mice reconstituted with bone marrow from ecSOD KO donors (KO -> KO) or C57Bl/6 mice with bone marrow from C57Bl/6 mice (WT -> WT). Experimental groups consisted of ecSOD KO mice reconstituted with bone marrow from C57Bl/6 mice (WT -> KO) or C57Bl/6 mice with bone marrow from ecSOD KO mice (KO -> WT). All mice were then infected with LM and evaluated for neutrophil recruitment and bacterial burden. We observed that ecSOD produced by hematopoietic cells leads to increased bacterial burden during LM infection, while ecSOD produced from somatic cells is essential for increased neutrophil recruitment. Collectively, our data suggest that ecSOD produced by both hematopoietic cells and somatic cells is involved in our observed phenomena; however, the contribution of ecSOD from each cell lineage is skewed towards either increased neutrophil recruitment or increased susceptibility to LM infection, but not both. These studies highlight the potential therapeutic value of ecSOD inhibitors to enhance immune responses during bacterial infections.Item EXTRACELLULAR SUPEROXIDE DISMUTASE ENHANCES NEUTROPHIL RECRUITMENT TO THE LIVER BY MODULATING THE EXTRACELLULAR ENVIRONMENT(2014-03) Witter, Alexandra R.; Break, Timothy J.; Indramohan, Mohanalaxmi; Berg, Rance E.Listeria monocytogenes (LM) infection represents one of the leading causes of death from foodborne infection, especially in immunocompromised individuals, and can cause spontaneous abortion in pregnant women; however, it is commonly used as a model to study the host immune response against infection. Our lab utilizes three groups of mice with varying levels of extracellular superoxide dismutase (ecSOD) activity (high, wild-type, and none) to determine the impact that this antioxidant enzyme has on the host immune response during LM infection. We have previously shown that ecSOD activity leads to decreased innate immune response against LM. Our current data shows that ecSOD activity protects the extracellular matrix from degradation, and leads to increased neutrophil recruitment; however, these neutrophils display inhibited function and therefore do not provide adequate protection against LM. This research is relevant due to the potential use of ecSOD inhibitors to enhance immune responses during bacterial infection. In addition, these findings help clarify the impact of the oxidative environment on the immune response and how antioxidants contribute to this dynamic. Purpose (a): Listeria monocytogenes (LM) is an intracellular foodborne pathogen that causes severe disease in immunocompromised individuals, spontaneous abortion in pregnant women, and results in ~25% mortality rate in infected individuals. Extracellular superoxide dismutase (ecSOD) converts superoxide into hydrogen peroxide in the extracellular milieu and protects against oxidative stress. We have previously shown that ecSOD activity inhibits innate immune responses during LM infection leading to increased bacterial burden; however, it is unclear whether ecSOD activity affects neutrophil recruitment and function in a cell-intrinsic manner or by modulating the extracellular environment. Methods (b): Congenic mice with high ecSOD activity (ecSOD HI), wild type ecSOD activity (ecSOD WT), or lacking ecSOD (ecSOD KO), on the C57Bl/6 background were used to perform adoptive transfer experiments after intravenous infection with ~10,000 wild-type LM (WTLM). Either isolated neutrophils or labeled whole bone marrow cells were transferred from ecSOD HI or ecSOD KO mice into ecSOD WT mice and then flow cytometry analysis was performed and colony forming units (CFUs) were calculated. Concentrations of hyaluronan and lymphotoxin alpha were determined by ELISA. Results (c): Whole bone marrow cell transfers indicated that there was no difference in recruitment of neutrophils transferred from ecSOD HI or ecSOD KO mice to the liver when the neutrophils were all in the same environment (ecSOD WT mice). In addition, neutrophils isolated from ecSOD HI or ecSOD KO mice showed no difference in their ability to protect against LM infection, as shown by equivalent CFUs, when in comparable environments (ecSOD WT mice). Analysis of hyaluronan concentrations – a component of the extracellular matrix (ECM) – indicated that ecSOD activity protects the ECM from degradation. Conclusions (d): We observed from adoptive transfer experiments that ecSOD activity does not affect neutrophil recruitment or function in a cell-intrinsic manner. Additionally, we determined that ecSOD activity protects the ECM, which is important for neutrophil trafficking. Overall, we concluded that ecSOD activity enhances neutrophil recruitment yet decreases their function by modulating the extracellular environment.Item EXTRACELLULAR SUPEROXIDE DISMUTASE ENHANCES NEUTROPHIL RECRUITMENT WHILE DECREASING THEIR FUNCTION(2013-04-12) Break, Timothy J.Purpose: Listeria monocytogenes (LM) causes spontaneous abortions in pregnant females and septicemia and meningitis in immunocompromised individuals, and results in ~25% mortality rate in infected individuals. Extracellular superoxide dismutase (ecSOD) converts superoxide into hydrogen peroxide in the extracellular milieu and protects against oxidative stress. The use of mice with varying levels of ecSOD serves as a novel approach to determine how an extracellular enzyme can impact immunity against an intracellular pathogen. Methods: Congenic mice with high ecSOD activity (ecSOD HI), wild type ecSOD activity (ecSOD WT), or lacking ecSOD (ecSOD KO), on the C57Bl/6 background, were infected with LM. Colony forming units (CFUs) were counted to determine bacterial load. Percentages of different cell types, cell-surface markers, and intracellular molecules (including ROS and proteins) were determined by flow cytometry. Neutrophil depletions were performed by i.p. injection of anti-Ly6G or isotype control antibody. Cytokine and chemokine concentrations were measured by ELISA. Results: EcSOD HI mice were more susceptible to LM infection than ecSOD WT and ecSOD KO mice. Interestingly, ecSOD HI mice have higher percentages of neutrophils in the liver compared to ecSOD KO mice, which was at least partially mediated by increased ecSOD-induced neutrophil-attracting chemokine production. Neutrophil depletions were performed, and ecSOD WT and KO livers had increased CFUs, while ecSOD HI mice showed slightly decreased CFUs, compared to isotype-treated mice. Furthermore, TNF-ɑ, which is important for clearance of LM, was highest in ecSOD KO mice. Interestingly, ecSOD activity decreased the ability of neutrophils to undergo oxidative burst, a mechanism to kill LM, and increased the amount of LM inside neutrophils. Lastly, ecSOD activity increased the percentage of myeloid-derived suppressor cells, which suppress immune responses. Conclusions: Our data show that ecSOD is detrimental during the early response to LM infection. An increased percentage of neutrophils in ecSOD HI livers, but no concurrent decrease in CFUs, suggests that ecSOD can impair the function of neutrophils. One way this may occur is through the internalization of ecSOD during phagocytosis, which decreases oxidative burst, allowing for LM to survive inside neutrophils. Furthermore, an increase in myeloid-derived suppressor cells in the ecSOD HI livers helps explain why these mice are more susceptible to infection.Item Extracellular Superoxide Dismutase Promotes Immature Neutrophil Egress from the Bone Marrow(2015-03) Witter, Alexandra R.; Break, Timothy J.; Indramohan, Mohanalaxmi; Mummert, Mark E.; Berg, Rance E.Extracellular superoxide dismutase (ecSOD) regulates extracellular concentrations of reactive oxygen species (ROS) to protect tissues during infection and inflammation. Using three groups of mice with varying levels of ecSOD activity, we have previously shown that ecSOD activity enhances neutrophil recruitment to the liver, yet inhibits the innate immune response against Listeria monocytogenes (LM) leading to increased host susceptibility. However, it is unclear whether ecSOD activity affects neutrophil recruitment and function in a cell-intrinsic manner or by modulating the extracellular environment. Using adoptive transfer experiments, we observed that ecSOD activity does not affect neutrophil recruitment or function in a cell-intrinsic manner. Additionally, we determined that ecSOD activity protects the extracellular matrix (ECM) and leads to an increase in phenotypically immature neutrophils in the bone marrow and liver. Collectively, our data suggest that ecSOD activity inhibits degradation of the ECM and promotes egress of immature neutrophils out of the bone marrow and into the liver where they provide inadequate protection against LM. These studies highlight the potential therapeutic value of ecSOD inhibitors to enhance immune responses during bacterial infections.Item FUNCTIONAL REGULATION OF PHAGOCYTIC CELLS BY IL-23 DURING LISTERIA MONOCYTOGENES INFECTION(2014-03) Indramohan, Mohanalaxmi; Break, Timothy J.; Witter, Alexandra R.; Berg, Rance E.Listeria monocytogenes (LM) is a Gram-positive intracellular pathogen that causes meningitis and septicemia in immunocompromised individuals, and spontaneous abortion in pregnant women. The versatility of LM makes it a useful tool for immunologists to understand how the immune system responds against harmful microorganisms. Cell recruitment mediated by the IL-23/IL-17 axis is important for protection against infectious diseases, but can cause damage during autoimmune disorders. By utilizing mice lacking IL-23 (IL-23p19 KO), our lab examines the role of this cytokine during a systemic bacterial infection. We have demonstrated that IL-23 promotes resistance against LM infection by increasing the recruitment of neutrophils to the liver, and monocytes to the spleen during LM infection. Interestingly, IL-23 or IL-17A is not required for enhancing phagocytic cell functions including phagocytosis, production of ROS, MPO, and pro-inflammatory mediators during LM infection. Understanding the significance of IL-23/IL-17axis in mediating the recruitment and function of immune cells will aid in the development of effective therapeutics depending on the disease condition. Purpose (a): Listeria monocytogenes (LM) is a Gram-positive intracellular foodborne pathogen that causes meningitis and septicemia in immunocompromised individuals, and spontaneous abortion in pregnant women. LM is widely used as a model pathogen to study host pathogen immune interactions. Cell recruitment mediated by the IL-23/IL-17 axis is necessary for protection against multiple infectious diseases, but can be detrimental during autoimmune disorders. We have previously shown utilizing mice lacking IL-23 (IL-23p19 KO) that IL-23 provides protection against LM infection by promoting the optimal recruitment of neutrophils to the liver, and monocytes to the spleen. The receptors for IL-23 and IL-17A are present on phagocytic cells including monocytes, neutrophils, and macrophages. However, it is not known whether IL-23 or IL-17A can impact the function of phagocytic cells during LM infection. Methods (b): Splenocytes and liver leukocytes were harvested from mice infected intravenously with ~10, 000 LM. Peritoneal wash was performed to isolate resident peritoneal macrophages. Flow cytometry was utilized to determine phagocytosis, production of reactive oxygen species (ROS), and myeloperoxidase (MPO). The concentrations of TNF-α, IL-1, IL-6, and nitric oxide (NO.) were measured by ELISAs/Griess assay. Results (c): Phagocytic cells isolated from control C57Bl/6 (B6) and IL-23p19 KO mice displayed equivalent phagocytic potential. There were no differences in the production of ROS or MPO from splenocytes isolated from both groups of mice. Furthermore, exogenous stimulation with rIL-23 or rIL-17A did not induce or enhance production of ROS or proinflammatory mediators from B6 splenocytes. Conclusions (d): IL-23 does not impact the function of phagocytic cells either by a direct or indirect mechanism during LM infection. Collectively, our data suggest that the lack of efficient recruitment of neutrophils to the liver, and monocytes to the spleen, results in a reduction in the overall levels of TNF-α and NO. and therefore, increases the susceptibility of IL-23p19 KO to LM infection.