Browsing by Subject "ecSOD"
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Item Allele-Specific Effects of Extracellular Superoxide Dismutase on Expression and Disease Susceptibility(2010-12-01) Jun, Sujung; Ladislav DoryOur lab previously reported a new allele for extracellular superoxide dismutase (ecSOD), expressed in 129P3/J mice (129), which differs from the wild-type, expressed in C57BL/6J and other strains. The newly discovered allele is associated with significantly increased circulating and heparin-releasable ecSOD activity and amount. To examine the properties of the two forms of ecSOD in an identical environment, I have generated congenic mice expressing either ecSOD allele on C57BL/6 genomic background. The congenic mice plasma ecSOD phenotypes show the same differences reported in the founder mice, indicating that the ecSOD genotype is largely responsible for the observed differences in the ecSOD phenotypes of the C57 and 129 strains. Tissue enzyme distribution of 129 allele is associated with higher levels of enzyme in most tissues; despite profoundly lower levels of the corresponding mRNA levels in the tissues. These results also suggest significant allele-specific differences in the regulation of ecSOD synthesis and intracellular processing/secretion of ecSOD. The increased rates of synthesis and secretion of 129 ecSOD relative to wt ecSOD is confirmed by using stably transfected CHO cells with either of ecSOD allele. The effects of the increased ecSOD levels in tissues on the susceptibility to asbestos-induced lung injury as well as bacterial infections were also investigated in congenic mice. Accordingly congenic mice with the 129 allele were significantly resistant to asbestos-induced fibrosis and injury. On the other hand, the expression of 129 allele significantly aggravated susceptibility to Listeria and Streptococcus infection compared to C57 allele and ecSOD KO mice, suggesting that ecSOD plays an important role in the modulation of immune responses triggered by bacterial infection. Overall this study confirmed the ecSOD allele-specific effects on the ecSOD phenotype and on the disease susceptibilities. In conclusion, the congenic mice offer an excellent model to examine the regulatory mechanisms of ecSOD expression and the role of ecSOD in various diseases involving oxidative stress.Item EXTRACELLULAR SUPEROXIDE DISMUTASE ENHANCES NEUTROPHIL RECRUITMENT WHILE DECREASING THEIR FUNCTION(2013-04-12) Break, TimothyPurpose: 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 Indirectly Enhances the Release of Immature Neutrophils from the Murine Bone Marrow(2016-08-01) Witter, Alexandra R.; Berg, Rance E.; Hodge, Lisa M.; Mummert, Mark E.Extracellular superoxide dismutase (ecSOD) regulates extracellular concentrations of reactive oxygen species (ROS) to protect tissues during infection and inflammation. Using ecSOD HI, ecSOD WT, and ecSOD KO mice, we have previously shown that ecSOD activity enhances neutrophil recruitment to the liver, yet inhibits the innate immune response against Listeria monocytogenes leading to increased host susceptibility. Using adoptive transfer experiments, we observed that ecSOD activity does not affect neutrophil recruitment or function in a cell-intrinsic manner. Additionally, we noted that ecSOD activity results in decreased retention of immature neutrophils in the bone marrow without altering granulopoiesis. Furthermore, we determined that ecSOD activity protects the extracellular matrix (ECM) and increases concentrations of neutrophil-attracting chemokines leading to an increase in immature neutrophils in the liver. Since ecSOD can be produced by cells from the hematopoietic lineage as well as non-hematopoietic cells, we used bone marrow chimeric mice to investigate the relative contribution of ecSOD produced by cells from each lineage. Ultimately, it was determined that ecSOD from both hematopoietic and non-hematopoietic cells contributes to the overall phenotype observed in ecSOD congenic mice. 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 L. monocytogenes. These studies highlight the potential therapeutic value of ecSOD inhibitors to enhance immune responses during bacterial infections.Item EXTRACELLULAR SUPEROXIDE DISMUTASE MODULATES THE ADAPTIVE IMMUNE RESPONSE DURING SECONDARY INTRACELLULAR BACTERIAL INFECTION(2013-04-12) Witter, AlexandraPurpose: 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, making it one of the leading causes of death from foodborne infection. Extracellular superoxide dismutase (ecSOD) converts superoxide into hydrogen peroxide in the extracellular milieu and protects against oxidative stress. While it has been shown that ecSOD decreases innate immune responses during LM infection, its role in a secondary infection model has not been explored. 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/OVA ΔActA and after 40 days challenged with LM/OVA. Colony forming units (CFUs) were counted to determine bacterial burden in the spleen and liver. Percentages of different cell types were determined by flow cytometry. IFN-𝛾 production and concentrations were determined by flow cytometry and ELISA respectively. Results: Our results indicate that ecSOD is protective in a secondary infection since ecSOD HI mice are better able to control bacterial burden than ecSOD KO mice with the ecSOD WT mice showing intermediate CFUs. The ecSOD KO mice exhibit a significantly lower percentage of dendritic cells and corresponding decreases in percentages of both overall CD8 T cells as well as memory CD8 T cells. There was also a decrease in percentages of overall CD4 T cells and memory CD4 T cells, primarily in the spleen. Additionally, there was a significant decrease in CD8 T cell specific IFN-𝛾 production in the spleen after overnight culture with both specific and non-specific stimulation. Conclusions: Our data indicate that ecSOD plays an important role in modulating cell-mediated adaptive immune responses during secondary intracellular bacterial infection. The combination of decreased dendritic cell and CD8 T cell populations, as well as decreased ability of CD8 T cells to produce IFN-𝛾, in ecSOD KO mice suggest that ecSOD may play a role in facilitating the activation of CD8 T cells and their ability to effectively respond during secondary LM infection.