Browsing by Subject "bacterial infection"
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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 THE ROLE OF IL-23 IN REGULATING THE FUNCTION OF PHAGOCYTIC CELLS DURING LISTERIA MONOCYTOGENES INFECTION(2013-04-12) Indramohan, MohanalaxmiPurpose: Listeria monocytogenes (LM) is a Gram-positive intracellular pathogen that causes meningitis and septicemia in immunocompromised individuals, and spontaneous abortion in pregnant women. IL-23 is a pro-inflammatory cytokine that promotes the recruitment of immune cells during multiple infectious and autoimmune diseases. Using IL-23p19 knockout (KO) mice we have demonstrated, that IL-23 is required for resistance against LM, and for the efficient recruitment of neutrophils to the liver, and monocytes to the spleen. However, it is not known whether IL-23 can impact the function of phagocytic cells including monocytes, neutrophils, and macrophages during LM infection. Methods: Mice lacking IL-23p19 (IL-23p19 KO) and C57BL/6 (B6) mice were infected with LM. For the isolation of thioglycollate-elicited peritoneal macrophages, mice were intraperitoneally injected with 1 ml of 2% thioglycollate, three days prior to harvest. Flow cytometry based assays were performed to measure phagocytosis and production of reactive oxygen species (ROS) using the dyes hydroethidine (HE) and 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA). ELISAs were performed to measure the amounts of TNF-ɑ and IL-6. Results: The phagocytic ability of neutrophils and monocytes in the spleen was equivalent between IL-23p19 KO and B6 mice. IL-23p19 KO mice had reduced monocyte-specific ROS production in spleen in comparison to B6 mice. However, the production of monocyte-specific or neutrophil-specific ROS was not induced or enhanced by the addition of recombinant IL-23 to B6 splenocytes. Interestingly, there was a reduction in the production of TNF-ɑ and IL-6 from thioglycollate-elicited peritoneal macrophages from IL-23p19 KO mice compared to B6 mice. Conclusions: IL-23 does not mediate phagocytosis by neutrophils and monocytes. Optimal production of monocyte-derived ROS requires IL-23 during LM infection. However, IL-23 does not directly mediate the production of ROS. Lastly, IL-23 is required for enhancing the production of TNF-ɑ and IL-6 from thioglycollate-elicited peritoneal macrophages.