Document Type

Dissertation

Date of Degree

Spring 2014

Degree Name

PhD (Doctor of Philosophy)

Degree In

Molecular and Cell Biology

First Advisor

Jessica G. Moreland

Abstract

Neutrophils, essential innate immune cells, recognize danger signals through receptors on their surface. Upon receptor ligation, neutrophils may undergo priming, a process involving limited reactive oxygen species (ROS) generation and partial degranulation. Priming facilitates neutrophil migration and prepares the cell for an enhanced response to a secondary stimulus, including a spike in ROS generation by NADPH oxidase 2 (NOX2). It is well established that NOX2-derived oxidants are involved in pathogen killing and that off-target effects can cause host tissue damage; however, several lines of recent evidence also support an anti-inflammatory function for NOX2 oxidants. First, patients with chronic granulomatous disease exhibit sterile inflammatory phenomena. Second, neutrophils lacking NOX2 function (genetically or pharmacologically) have an inflammatory phenotype under resting conditions. Finally, NOX2-deficient mice exhibit enhanced localized inflammation in several disease models. The goals of this thesis were to investigate an anti-inflammatory function for NOX2 during systemic inflammation and to further elucidate mechanisms of neutrophil priming, with particular focus on priming through Toll-like receptor 2 (TLR2). Using a murine model of sterile systemic inflammatory response syndrome (SIRS), we observed that NOX2-deficient mice had dramatically increased mortality compared to WT mice. While both genotypes developed SIRS, characterized by hypothermia, hypotension, and leukopenia, the WT mice recovered within 48 h whereas the NOX2-deficient mice did not. Moreover, NOX2 function limited the extent of pulmonary pathology as significant lung injury was noted in the NOX2-deficient mice compared to the WT mice. Plasma analysis revealed that several inflammatory cytokines were persistently elevated in the NOX2-deficient mice, likely contributing to the ongoing inflammatory response. One of the complications seen in human SIRS patients is the development of multiple organ dysfunction syndrome (MODS). Thus, we next investigated the role of NOX2 in the progression from SIRS to MODS. Cellular analysis revealed continued neutrophil recruitment to the peritoneum and lungs of the NOX2-deficient mice and altered activation states of both neutrophils and macrophages. Histology showed multiple organ pathology indicative of MODS in the NOX2-deficient mice, and several inflammatory cytokines were elevated in lungs of the NOX2-deficient mice. Overall, these data suggest that NOX2 function protects against the development of MODS and is required for normal resolution of systemic inflammation. As we utilized a TLR2/6 agonist (zymosan) to induce SIRS in our in vivo model, we wanted to investigate neutrophil priming through TLR2 in an in vitro model. Notably, we determined that a TLR2/6 agonist, FSL-1, primed neutrophils from all donors to a similar extent, evidenced by direct and primed ROS generation, MAPK signaling, limited degranulation, and cytokine secretion. Surprisingly, Pam3CSK4, a TLR2/1 agonist, primed neutrophils from a subset of donors to a much greater extent than neutrophils from other donors. We demonstrated that the different neutrophil priming responses were the consequence of a common TLR1 polymorphism. In sum, the data presented here significantly advance our understanding of the roles of NOX2 and TLR2 signaling in host inflammation and neutrophil priming. This research could advance the development of therapies that target pathogenic neutrophil subsets in inflammatory conditions without compromising innate immune function

Keywords

Inflammation, NADPH oxidase, Neutrophil, SIRS, Toll-like receptor

Pages

xv, 170 pages

Bibliography

Includes bibliographical references (pages 153-170).

Copyright

Copyright 2014 Laura Christine Whitmore

Included in

Cell Biology Commons

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